RNA Processing: A Practical Approach: Volume II

RNA Processing ,\ Practical Approach \'OJ.l i:\IE II

Edited by S. J. HI GGINS a nd B. D. HAM ES

7

u

l1

t

mG

•GTP

3'

5'

I

(I

I + *G '011

AAUAAA

r-(A)0

~

- -

RNA Processing Voluine II A Practical Approach Edited by STE P H E N J. HIG GI N S and B . DA VI D H A ME S Department of Biochemistry and Molecular Biology, University of Leeds

-atOXFORD l'XT\'ERSITY PRESS

Oxford :'lit:w York Tokyo

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~t

The Practical Approach Series SERIES EDITORS

D. RICKWOOD Deporrment of B1ology, Uni~·ersuy of Essex Wn·enhoe Pork, Colchester, Essex C04 JSQ, UK

B. D. HAMES Deportment of Biochemistr> and ..,1oleculor Btolog; Unn·ersity of Leeds, Leeds LS2 9JT, UK

Affinity Chromatography Anaerobic Microbiology Animal Cell Culture (2nd edition) Animal Virus Pathogenesis Antibodies I and II Behavioural Neuroscience Biochemical Toxicology Biological Data Analysis Biological Membranes Biomechanics-Materials Biomechanics-Structures and Systems Biosensors Carbohydrate Analysis Cell-Cell Interactions The Cell Cycle Cell Growth and Division Cellular Calcium Cellular Interactions in Development Cellular Neurobiology Centrifugation (2nd edition) Clinical Immunology Computers in Microbiology Crystallization of Nucleic Acids and Proteins Cytokines The Cytoskeleton Diagnostic Molecular Pathology I and II Directed \.iutagenesis

DNA Cloning I, 11, and £II Drosophila Electron Microscopy m Biology Electron Microscopy in Molecular Biology Electrophysiology Enzyme Assays Essential Developmental Biology Essential Molecular Biology I and II Experimental Neuroanatomy Fermentation Flow Cytometry Gas Chromatography Gel Electrophoresis of Nucleic Acids (2nd edition) Gel Electrophoresis of Proteins (2nd edition) Gene Targeting Gene Transcription Genome Analysis Glycobiology Growth Factors Haemopoiesis Histocompatibility Testing HPLC of Macromolecules HPLC of Small Molecules Human Cytogenetics r and II (2nd edition) Human Genetic Disease Analysis

Immobilized Cells and Enzymes Immunocytochemistry In Situ Hybridization Iodinated Density Gradient Media Light \ticroscopy in Biolog} Lipid Analysis Lipid todification of Proteins Lipoprotein Analysis Liposomes Lymphocytes Mammalian Cell Biotechnology \1ammalian Development !\1edical Bacteriology !\1edical Mycology !\1icro..:omputers in Biochemistry Microcomputers in Biology Microcomputers in Physiology Mitochondria Molecular Genetic Analysis of Populations Molecular Imaging in Neuroscience Molecular Neurobiology ~tolecular Plant Pathology I and li Molecular Virology Monitoring Neuronal Activity Mutagenicity Testing "leural Transplantation Neurochemistry l'ieuronal Cell Lines NMR of Biological Macromolecules Nucleic. Acid and Protein Sequence Analysis Nucleic Acid Hybridisation Nucleic Acids Sequencing Oligonucleotides and Analogues Oligonucleotide Synthesis PCR Peptide Hormone Action

Peptide Hormone Secretion Photosynthesis: Energy Transduction Plant Cell Culture Plant Molecular Biology Plasmids (2nd edition) Pollination Ecolog:r Postimplantation 1\lammalian Embryos Preparative Centrifugation Prostaglandins and Related Substances Protein Architecture Protein Engineering Protein Function Protein Phosphorylation Protein Purification Applications Protein Purification Methods Protein Sequencing Protein Structure Protein Targeting Proteolytic Enzymes Radioisotopes in Biology Receptor Biochemistry Receptor-Effector Coupling Receptor-Ligand Interactions Ribosomes and Protein Synthesis RNA Processing Signal Transduction Solid Phase Peptide Synthesis Spectrophotometry and Spectrofluorimetry Steroid Hormones Teratocarcinomas and Embryonic Stem Cells Transcription Factors Transcription and Translation Tumour lmmunobiology Virolog) Yeast

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A Pract1ca/ Approach is a regtstered trade mark of the Chancellor, \fosters, and Scholars of the Unmm1ty of Oxford trading as Oxford Universtty Press Published in the United States by Oxford Um>oersuy Press, New York @Ox/i>rd Uniwrw\ Press. /994 All rights reserved. !\'o part of thts pub/teal/on may be reproduced, stored m a reme•·a/ system. or transmmed, in any form or by any means, >wthout the prior permiSSIOn In wrtllng of Oxford Unll•ersity Press. Within the UK, exceptions are allowed in respect of any fatr dealing for the purpose of research or private study, or criticism or rewew, as permuted under the Copyright, Designs and Patents Act, 1988, or in the case of reprographic reproduction in accordance w1th the terms of l1cences issued by the Copyright Licensmg Agency. Enquiries concerning reproduction outside those terms and m other countries should be sent to the Rtghts Department, Oxford Uni,·erslty Press, at the address above. This book 1s sold subJect to the conditton that 11 shall not, by way of trade or otherwise, be lelll, re-sold, hired out, or otherwise ctrculated wuhout the publisher's prior consent in an; form of binding or cover other than that In which 11 ts published and without a similar condition including this condition being tmposed on the subsequent purchaser. Users of books tn the Practical Approach Series are advised that prudent laboratory safety procedures should be followed at all times. Oxford Universuy Press makes no representation, express or 11nplied, m respect of the accuracy of the material set forth In books in tl11s sertes and cannot accept any legal responsibility or liability for any errors or otmssions that may be made. A catalogue record for tillS book is available from the Brittsh Library Ltbrary of Congress Cataloging m Publication Data RNA processmg: o practical approach edited by Stephen J. H1ggins and B. David Hames. p. cm.-(The Practical approach series, /35, 136) Includes b1blwgraphical references and mdex. I. RI\A-Research-l..oborotory manuals. 1. GenetiC regulotionResearch-I.Aborotory manuals. I. Higgins, S. J. (Ste,·e J.J f/ Homes, 8. D. Iff. Series QP623.R58 !993 574.87'3283-dclO 93-13019 ISBN 0 19 963344 4 (hlb) ISBN 0 19 963343 6 (plb) ISBN 0 19 963471 8 fhlb) ISBN 0 /9 963470 X (p/b)

J Volume I

J

Volume II

Typeset by Dobb1e Typesetlmg Ltmued, Tavwock, Devon Prmted and bound m Great Bmom by Information Press, Ltd.. Eynsham, Oxon

Foreword RNA processing: two helpful guides for cutting , pasting, trimming , and editing RNA TOM MANIATIS

During the past decade, the study of RNA processing has risen to the forefront of molecular biology. At one time, the RNA processing field was a relatively small group of investigators interested in the trimming and packaging of prokaryotic transfer RNA and ribosomal RNAs. However, with the discovery of a bewildering array of RNA processing events in both prokaryotes and eukaryotes, the field has become a burgeoning enterprise. This change is reflected in the annual RNA processing meeting which has been rapidly transformed from a small informal gathering to one bursting the seams of the expanded meeting facilities at the Cold Spring Harbor Laboratory. Due to the pace of this unprecedented growth in size and diversity, no one has stopped long enough to compile a detailed description of even the most basic techniques used to study RNA. Students in the field are therefore introduced to the laboratory with handwritten protocols passed from person to person. The methods compiled in this book and its companion volume by leaders in the field should therefore contribute significantly to the training of new students and hopefully lead to the development of new techniques. Volumes I and II of RNA processing: a practical approach cover virtually all aspects of RNA processing, including capping, splicing of both pre-mRNA and tRNA, polyadenylation, editing and ribosomal RNA processing. In addition, an overvtew of ribozymes is provided. An understanding of the various types of RNA processing in vivo is an essential prerequisite for studying in vitro the mechanisms involved . Therefore, the description of methods for RNA mapping, including nuclease Sl mapping, primer extension analysis, and the use of the polymerase chain reaction should be very useful. An important technical advance in the analysis of RNA processing in vitro was the development of plasmid vectors containing bacteriophage-specific promoters for synthesizing labelled substrate RNAs. A description of the Synthesis and purification of in vitro transcripts produced with SP6, T7, and polymerase is therefore an essential part of the coverage. Also important lS a description of the preparation and optimization of nuclear extracts for several

!3

Foreword types of in vitro RNA processing, 3 '-end formation and polyadenylation. The manipulation and fractionation of these extracts have led to the purification of individual processing components and the cloning and characterization of the corresponding genes. Methods have also been developed for the selective inactivation of individual snRNPs using specific DNA oligonucleotides, or the depletion or punfication of )nRNP particles using oligo(2' -0-alkylribonucleotides). In addition, immunoaffin.ity purification methods for purifying snRNPs are presented as well as procedures for reconstituting individual snRNPs from purified proteins and RNAs. These tools for characterizing snRNPs will become increasingly important as the details of protein-protein and protein-RNA interactions in the spliceosome are unravelled . An area less intensely investigated but of increasing importance, is the turnover of mRNA. There are now many examples in which gene expression during development is regulated by the selective turnover of specific RNAs. In add:ition, it is now clear that the rapid decrease in the expression of cyrokines after induction by extracellular inducers involves a specific recognition sequence in the 3 non-coding sequence of mRNA. The chapter describing methods for studying mRNA turnover in vitro will stimulate further studies of this important problem. The most recently discovered and most unexpected forms of RNA processing are autocatalytic splicing and RNA editing. The discovery of group I intron self-splicing and the RNase P ribozyme has led rapidly to detailed characterization of the catalytic reactions involved. Dissection and manipulation of the group I intron ribozyme have led to the creation of new catalytic activities and an understanding of the role of RNA secondary and tertiary structure in ribozyme function . Similarly, the elucidation of the structure and activity of group li introns has provided important new insights into the nature of ribozymes and has led to critical insights into the mechanisms involved in pre-mRNA splicing. Both group 11 intron and pre-mRNA splicing proceed through a similar branched RNA intermediate. Although the former process is autocatalytic and the latter require~s multiple components assembled into a spliceosome, the catalytic events may be quite similar. In fact, the parallels between the role of RNA structure in group II intron splicing and the role of snRNA-pre-mR'JA interactions have increased by the recent demonstration of dynamic interacuons between snRI'.JA editing Introduction Preparation of mitochondrial extracts Terminal uridylyl transferase (TUTase) RNA ligase Cryptic R ase gR A:mRNA ch•maera-forrning activity

96 96

97 97 99 100 101

Acknowledgement

103

References

I03

4. Analysis of messenger RNA turnover in cell-free

extracts from mammalian cells

107

Jeff Ross 1. Introduction

107

2. Choice of mRl'JA substrate

I08 I09 J J0

Endogenous substrates (free mR P or polysome-associated mRNP) Exogenous substrates

3. Preparation of undegraded mRNA and polysomes

110

4. Preparation of cell extracts for in vitro mRNA decay reactions

III

5. Methods for performing in vitro mRNA decay reactions

119 119 123 125

Detergent-free extracts Lysolecithin extracts Reticulocyte translation extracts

6. mRNA detection, data interpretation, and troubleshooting mRNA detection and some typical in vitro mRNA decay experiments Data interpretation Troubleshooting

References

126 126 130 131 132

5. Ribosomal RNA processing in vertebrates

135

Cathleen Enright and Barbara Sol/ner- Webb 1. Introduction

135

2. Analysis of rR.'JA processing in vivo

137 137

Introduction Analysis of rRNA processing by hybrid selection and gel electrophoresis of cellular rRNAs Use of psoralen cross-linking for the analysis of associations bel\veen rRNAs in vivo Electron microscope analysis of rRNA synthesis (Miller spreading) XV

139 142 143

Contents

3. Analysis of rRNA processing in \'itro

144

lmroducuon Preparation of synthetic rR!-.A ~ubstrates Preparation and use of mouse S-100 processing extract Preparation and use of nucleolar and cytoplasmic processing extracts from HeLa cells Cleavage of pre-rRNA using purified nucleolar endonuclease Use of deletion analysis in the investigation of rRNA processing

144 144 148

4. Analysis of protein-RNA associations in rRNA processing Gel-shift analysis Analysis of rRNA associations by rate zonal centrifugation Use of UV cross-linking to detect polypeptides associated with rRNA in processing complexes

155 157 157

5. Assessment of the requirement for cellular R.J"'JA in rRNA processing Micrococcal nuclease digestion Oligonucleoude-directed RNase H digesuon of cellular R A~ in\olved m pre-rRNA processing Immunological analysis of polypeptides required for processing

150 155 155

160

164 164 165 169

6. rRNA processing associated with transcriptional termination 169 3' end-processing in the mouse 3' end-processing in Xenopus laevis

170 170

Acknowledgements

170

References

170

6. Processing of transfer RNA precursors

173

Chris L. Greer 1 . Introduction General methods tRNA structure and function Overview of processing pathways Biochemical analysis of tRNA proce~sing

173 173 173 175 176

2. Preparation of pre-tRNA processing substrates

176 176 176

Introduction Preparation of nuclease-free carriers Preparation of tRNA substrates from whole cells Preparation of substrates by in vitro transcription

3. Preparation of tRNA processing extracts Preparation of crude ceLl extracts Partial purification of processing activities Preparation of highly-purified processing components

xvi

177 181 185 187 189 191

Contents 4. Pre-tRNA processing assays Strategy Assay of pre-tRJ'IA splicing As a:r of base modifications and tR:-IA splice junctions

195 195 196 199

Acknowledgements

206

References

206

7 . Ribozymes

211

David A. Shub, Craig L. Peebles, and Arnold Hampel 1. Introduction

211

2. Group 1 intron ribozymes The self-splicing activity of group I introns Detection of self-splicing introns in cellular RNA Confirmation of self-splicing by in vitro transcription of cloned DNA containing putative group I introns

211 211 213

3. Group II intron ribozymes Identification of group l1 introns Group II intron secondary structure and functional anatomy Potential of group II intron ribozymes for practical use Self-splicing of group l1 introns Other reactions of group 11 intron ribozymes

217

217 219 220

4. Hammerhead and hairpin ribozymes

230

215

221

224

Introduction 230 Applications of hammerhead and hairpin ribozymes in molecular biology 232 Use of hammerhead and hairpin ribozymes to target specific RNA transcripts 232 Use of autocatalytic cassettes to generate transcripts with defined 5' or 3' termini 236

Acknowledgements

237

References

237

Appendix: Suppliers of specialist items Contents of Volume I Index

xvii

241

245 247

Contributors BEAT BLUM Department of Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland. CATHLEEN ENRIGHT Department of Biological Chemistry, School of Medicine, The Johns Hopkins University, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA. Y AS UH lR O FURUICH I Biotechnology Subdivision, Nippon Roche K.K., Nippon Roche Research Center, 200 Kajiwara, Kamakura-Shi, Kanagawa 247, Japan. CHRIS L. GREER Department of Biological Chemistry, College of Medicine, D240, Med Sci I, University of California, Irvine, CA 92717, USA. ARNOLD HAMPEL Department of Biological Sciences, Northern Illinois University, De Kalb, Illinois 60115-2861 , USA. B. DAVID HAMES Department of Biochemistry and Molecular Biology, University of Let!ds, Leeds LS2 9JT. STEPHEN J. HIGGINS Department of Biochemistry and Molecular Biology, University of Let!ds, Leeds LS2 9JT. WALTER KELLER Abteilung Zellbiologie, Biozentrum der Universitat Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. TOM MANIA TIS Department of Biochemistry and Molecular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA. CRA IG L. PEEBLES Department of Biological Sciences, 365 Crawford Hall, University of Pittsburgh, Pittsburgh, P A 15260, USA. JEFF ROSS McArdle Laboratory for Cancer Research, University of Wisconsin··Madison , 1400 University Avenue, Madison , Wisconsin 53706, USA.

Contributors DAVID A . SHUB

Center for '\ltolecular Genetics, Depanmem of Biological Sciences 126, State University of New York at Albany, 1400 Washington Avenue, Albany NY 12222, USA . AARON J. SHATKlN

Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, Ne,-.. Jersey, NJ 08854-5638, USA. LARRY SIMPSON

Howard Hughes Medical Institute, Department of Biology, University of California, Los Angeles, 10833 Le Coate Avenue, Los Angeles, California, CA 90024-1662, USA. AGDA M. SIMPSON

Department of Biology, University of California, Los Angeles, 405 Hilgard Avenue, Los Angeles, California, CA 90024-1606, USA. BARBARA SOLLNER-WEBB

Department of Biological Chemistry, School of Medicine, The Johns Hopkins University, 725 Nonh Wolfe Street, Baltimore, MD 21205-2185, USA. ELMAR WAHLE

Abteilung Zellbiologie, Biozentrum der Universitat Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.

XX

Abbreviations A Am

A260 A600 AdoHey AdoMet AMP ASV ATP bp BHI BHK BSA

c

eDNA Ci

CIP CMP c. p.m.

CPSF CPV

CTP D DAPI dATP ddATP DEAE DBAE

DEPC DHFR DMEM DMSO DNase d. p.m.

DTE DTT EBS EDTA

EGTA

adenine/ adenosine/ adenylate absorbance (259 nm) absorbance (260 nm) absorbance (600 nm) S-adenosylhomocysteine S-adenosylmethionine adenosine 5 1 -monophosphate avian sarcoma virus adenosine 5 1 -triphosphate base pair(s) brain heart infusion baby hamster kidney bovine serum albumin cytosine/ cytidine/ cytidylate complementary DNA curie calf intestinal alkaline phosphatase cytidine 5 1 -monophosphate counts pe1r minute cleavage and polyadenylation specificity factor cytoplasmic polyhedrosis virus cytidine 5 1 -triphosphate dihydrouridine 4 1 ,6 1 -diamidino-2-phenylindole deoxyadernosine 5' -triphosphate dideoxyadenosine 5 1 -triphosphate diethyl aminoethyl dihydroxyboryl aminoethyl diethyl pyrocarbonate dihydrofolate reductase Dulbecco's modified Eagle's medium dimethyl sulphoxide deoxyribonuclease disintegrations per minute dithioerythritol dithiothreit.ol exon binding sequence ethylenediamine-tetraacetic acid ethylene glycol-0 1 0 1 -bis(2-aminoethyi)-N,N,N 1 ,N 1 -tetraacetic acid

Abbreviations elF ETS

FPLC g

G

GMP gRNA

GTP h HGPRT

HIV HPLC 1BS ig

IPTG ITS kapp

kbp kcat

kDa kDNA Km kRNA

LDH rn 1A

m6A

m3G m7G min mRNA mRNP

nt

NADH NDK

NES NTP ori

PABP

PAGE

PBS PCR

PCV

PEG PEl

eukaryotic initiation factor external transcribed spacer fast-performance liquid chromatography gravit) guanine/ guanosine/ guanylate guanosine 5 1 -monophosphate guide RNA guanosine 5 1 -triphosphate hour(s) hypoxanthine-guanine phosphoribosyltransferase human immunodeficiency virus high-performance liquid chromatography intron binding sequence internal guide (sequence) isopropyi-{3-D-thiogalactoside internal transcribed spacer apparent equilibrium constant kilobase pair(s) turnover number kilodaltons kinetoplast DNA Michaelis constant kinetoplast RNA lactate dehydrogenase !-methyl adenine 6-methyl adenine 2,2, 7-trimethyl guanosine 7-methyl guanine minute(s) messenger RNA messenger ribonucleoprotein (particle) nucleotide(s) reduced nicotinamide dinucleotide nucleoside diphosphate kinase sodium acetate, EDTA, SDS buffer nucleoside 5 1 -triphosphate origin of replication poly(A)-binding protein polyacrylamide gel electrophoresis phosphate-buffered saline polymerase chain reaction packed cell volume polyethylene glycol polyethylenimine xxii

Abbre\·iations phosphoenolpyruvate pre-edited region inorganic phosphate pyru\'ate kinase PK phenylmethylsulphonyl fluoride PMSF packed nuclear volume PNV pans per million p.p.m. pounds per square inch p.s.i. polyvinyl alcohol PYA polyadenylic acid Poly(A) Poly(A)-RNA polyadenylated RNA polycytidylic acid Poly(C) ribonuclease RNase ribonucleoprotein (particle) RNP ribonucleoside 5 ' -triphosphate rNTP revolutions per minute r.p.m. ribosomal RNA rRNA Renografin, sucrose, EDTA buffer RSE Renografin, sucrose, Tris, EDTA buffer RSTE ribosomal salt wash RSW s svedberg SDS sodium dodecyl sulphate SDS-PAGE SDS-polyacrylarnide gel electrophoresis sec second(s) SER spliced exon reopening SET salt, EDTA, Tris buffer SJH splice junction hydrolysis snRNA small nuclear RNA snRNP small nuclear ribonucleoprotein (particle) sse standard saline citrate STE sucrose, Tris, EDTA buffer STM sucrose, Tris, MgC1 2 buffer SV40 simian virus 40 T ribothymidioe/ ribothymidylate TAE Tris-acetate-EDTA buffer TBE Tris-borate-EDTA buffer TCA trichloroacetic acid TE Tris-EDTA buffer TEAB triethylammoojum bicarbonate TEMED N,N,N ',N '-tetramethylenediamine TLC thin layer chromatography Tm melting temperature TMA tetramethylammonium chloride TMN Tris, MgCI 2, NaCI buffer

PEP PER P;

xxili

Abbreviations TMY tRNA TS TSE TUTase

u UDP UMP UTP

uv

v V;

v,

w X-gal it

tobacco mosaic virus transfer RNA Tris saline Tris, SDS, EDTA buffer terminal uridylyl transferase uracil!uridine/ uridylate uri dine 5' -diphosphate uridine 5 1 -mono phosphate uridine 5 1 -triphosphate ultraviolet volt(s) initial velocity resuspension volume watt(s) 5-bromo-4-chloro-3-indolyl-~-o-galactoside

pseudouridine

:xxiv

3' end-processing of m.RNA ELMAR WAHLE and WALTER KELLER

1 . Introduction: overview of 3 ' end-processing in

mammalian cells The 3 1 -end of a eukaryotic messenger RNA is not formed by precise termination of transcription. Rather, RNA polymerase II synthesizes a precursor RNA extending far beyond the 3 1 -end of the mature mRNA. The end of the mature RNA is then generated by post-transcriptional processing of the precursor RNA. Formation of the 3 1 -end is an essential step in mRNA synthesis; mutations in the RNA sequences that direct this process lead to a strongly reduced steady-state level of mRNA. In mammalian cells, almost all mRNAs receive their 3 1 -ends in a process consisting of two reactions: (a) The precursor RNA is first cleaved endonucleolytically at a particular site 3 1 (downstream) of the coding sequences. 1 (b) The 5 (upstream) fragment is then extended by the addition of approximately 200 adenylate residues (po1yadeny1ation), whereas the downstream fragment is degraded. The reactions depend on two sequences in the precursor RNA: the highly conserved sequence AAUAAA 10-30 nt upstream of the cleavage site and one or more sequence elements within approximately 50 nt downstream of this site. The downstream elements are not well defined; they may be either GU-rich or runs of U. In yeasts and plants, mRNAs are also polyadenylated. However, the sequence requirements and, possibly, mechanisms of 3 ' -end formation are different from those outlined above (for a recent review, see ref. 1). Cleavage and polyadenylation can be carried out in vitro in a nuclear extract from tissue culture cells (2). A complex sex of protein factors has been identified that catalyses both reactions in a tightly coupled fashion. At least two of these Proteins, and possibly more, are 'cleavage factors' involved only in the initial endonucleolytic scission; they are dispensable for polyadenylation. Two other factors are also required for the cleavage reaction but, in addition, carry out Polyadenylation. These are poly(A) polymerase, the enzyme that catalyses the Polymerization of the poly(A) tail from ATP, and a factor that binds to

1: 3' end-processing

of mRNA

AAUAAA and provides the polymerase with its specificity for RNAs containing this sequence. This latter factor has been described under different names; the authors will refer to it as cleavage and polyadenylation specificity factor (CPSF). In addition, a poly(A)-binding protein is involved in the elongation phase of poly(A) tail synthesis. For recent reviews, see refs I, 3. In mammalian cells, the only kno.... n exceptions to the mRNA processing pathway described above are the precursors to the major histone mRNAs. These undergo an endonucleolytic cleavage just downstream of a hairpin-loop structure. Poly{A) tails are not added. The reaction can be analysed in vitro (4). One of the factors required is the U7 snRNP. \1essenger RNA sequences involved in this form of 3' end-processing include a region which basepairs with the U7 RNA. The other factors involved in the reaction have nor been well characterized. For a review, see ref. 5. This chapter describes methods used to study endonucleolytic cleavage and polyadenylation of mRNA precursors in vitro. Procedures that are of basic importance and general applicability have been included. References will be given for procedures which are beyond the scope of this chapter or with which the authors have no personal experience. Section 2 deals with 3 ' end-processing in vitro in nuclear extracts from HeLa cells. Section 3 describes methods for the purification and assay of processing factors. Section 4 gives ad"ice on monitoring processing in vivo.

2. 3'-end cleavage and polyadenylation in

nuclear extracts 2.1 Introduction Processing of the 3 '-end of an RNA transcript may be studied in nuclear extracts with the use of appropriate RNA substrates synthesized in vitro. This section describes the preparation of nuclear extracts active in 3 ' end-processing and their use in studies of the formation of polyadenylation complexes, as well as of the cleavage and polyadenylation reactions themselves.

2.2 RNA substrates for 3' end-processing in vitro In vilro. RNA processing reactions are most easily analysed by the use of radiolabelled RNA substrates. These precursors are synthesized generally by 'run-ofr transcription of appropriate gene sequences cloned downstream from promoters for bacteriophage-encoded RNA polymerases (e.g. T3. T7, or SP6 polymerases). Protocols for the synthesis of RNA substrates and their purification by denaturing polyacrylamide gel electrophoresis are described in Volume I, Chapter I (Section 2). The following practical points should be observed when RNA substrates for m vitro processing are synthesized: 2

Elmar Wahle and Walter Keller (a} There is some suggestion that the 5' -cap structure of an RNA transcript plays a role in 3 ' end--processing, though this is still controversiaL The cap certainly stabilizes the RNA during the incubation in nuclear extracts. In general, capping of the RNA substrate is, therefore, desirable. (Volume I, Chapter I, Section 2.2 provides a protocol for the synthesis of capped RNA substrates.) (b) RNA substrates of different specific radioactivities are required for different assays (see Section 2.5). Make RNA of the desired specific radioactivity by adjusting the ratio of labelled to unJabelled UTP in the transcription reaction (see Volume I, Chapter I, Section 2.2). (c) In vitro processing reactions shouJd be carried out with known quantities of the RNA substrate. Calculate the concentration of radioactive RNA synthesized from the amount of radioactivity incorporated (count an aliquot of the RNA preparation in a scintillation counter), the specific radioactivity of the [ 32P] UTP used in the transcription reaction, and the number of UMP residues in the RNA. Further advice is given in Volume I, Chapter I, Section 2.3.4. (d) Store the RNA as an ethanol precipitate at - 20 °C. Brief vortexing of this should be sufficient to generate a homogeneous suspension so that the desired quantity of RNA for use in processing reactions can be withdrawn. If a homogeneous suspension cannot be generated for accurate and reproducible sampling, precipitate the RNA (see Protocol 5, steps 1-3), dissolve the pellet either in water or 5007o ethanol, and store it at - 20 oc. In these cases, lyophilize an aliquot of the RNA prior to use. (e) If the RNA is to be used as a 'precleaved' substrate for polyadenylation (see Section 2.5.4), a free hydroxyl group at the 3'-end is essential. Both chemical hydrolysis of RNA and degradation by the majority of nucleases leave a phosphate group at the 3 '-end which prevents polyadenylation. While RNA synthesized by the standard run-off procedure is generally a good substrate for polyadenylation, the authors have encountered phage T7 RNA polymerase from one supplier which generated RNA that could not be extended by poly(A) polymerase. The quality of the RNA 3 ' -ends can be checked by a preliminary nonspecific polyadenylation procedure. For this purpose, Mnh -dependent extension with unlabelled ATP followed by gel electrophoresis (see Section 2.5.5) shouJd be used for radiolabelled RNA. Ext.ension with radiolabelled A TP followed by TCA precipitation (see Protocol 8) should be used for unlabelle-d RNA substrates. Many studies of 3 end-processing in vitro have used two particular polyadenylation sites: the site in SV40 late mRNA and the L3 site of adenovirus 2. 3

1: 3' end-processing of mRNA If a polyadenylation site is to be used that has not been characterized before, the following points should be noted:

(a) Use a genomic fragment rather than a eDNA fragment to generate the RNA substrate since the essential downstream elements will not be present in the latter. Design the run-off transcription so that at least 50 nt downstream of the polyadenylation site are included in the precursor RNA. (b) Some genes contain additional 3' end-processing signals upstream of the

AAUAAA sequence. Therefore, include in the RNA substrate about 150 nt upstream of this sequence. (c) To test whether any processing event observed is genuine, use an RNA precursor with a point mutation in the AAUAAA sequence (e.g. AAGAAA) as a control.

2.3 Preparation of nuclear extracts for 3' end-processing Because the substrate for most RNA processing assays is a small quantity of radiolabelled RNA, RNases pose a major problem. In practice, the high abundance of RNases in extracts of many tissues limits the choice of starting material for extract preparation to cultured cell lines. Nuclear extracts from HeLa cells are the standard material for the analysis of RNA processing in vitro because they are low in endogenous RNases and can be readily prepared. The following procedure (Protocol 1) uses freshly-harvested HeLa cells for the preparation of nuclear extract, but it also works with cells that have been frozen. However, fresh cells swell better in the hypotonic buffer and thus can be more easily lysed. The use of a Dounce homogenizer for cell lysis requires a certain minimal volume (approximately 2 ml for a small homogenizer). If small amounts of cells are to be processed, they can be taken up in a syringe and forced repeatedly through a 25-gauge needle (7). Alternatively, cells are not broken mechanically but lysed by the addition of lysolecithin (8). Usually, the nuclear extract is dialysed before use since the salt concentration limits the amount of non-dialysed extract that can be added to a processing reaction. However, the non-dialysed extract seems to be more active in the cleavage assay (see Protocol 6 and Section 2.5.3). Note that the buffers used to prepare the nuclear extract (see Protocol/) contain 1.5 mM Mg2 +, whereas dialysis buffer (buffer D; see Protocol 1) does not. The amount of Mg2 • added to in vitro reactions may thus have to be adjusted. Although Protocol I was developed for use with HeLa cells, extractS from other cell lines can generally be made by the same procedure. However, the protocol cannot be applied to the preparation of nuclear extract from solid tissue (e.f. calf thymus, see Section 3.2.3). 4

Elmar \Vahle and \\'alter Keller Protocol 1. Preparation of Hela cell nuclear extract for 3 ' end-processinga Equipment and reagents • Oounce homogen1zer lcapac1ty 120 ml for 151itres of Hela ce ll culture) with a type S pestle

• H1gh-salt buffer C (buffer C contaimng 1.2 M NaCI)

• Phase-contrast m1crosgenate to a beaker in an ice bath and sttr 1t gently for 30 min on a mag netic st1rrer. 10 . Centrifuge the ho,mogenate for 30min at 16000 x gmax·

Continued

5

1: 3' end-processing o.f mRl\'A Protocol 1. Continued 11. Remove the supernatant (nuclear extract) D1alyse (optional) the extract for 5 h against 2 litres of buHer D

12 . Freeze the nuclear extract 10 suttable aliquots 1n liquid nitrogen and store them at -70 °C. ' A mod1flcat1on of the ong1nal protocol of Dignam et a/. (6) developed by Robin Reed (Harvard Medical School! and communicated to the authors by Ryszard Kole (University of North Carolina). • All buffers can be stored for several weeks in a refrigerator. • Adjust a 1 .0 M stock solution of this compound at room temperature to the pH indicated. Use this stock to make up the buffer without further adjustment of the pH. d Add OTT just prior to use from the 1.0 M stock solution. • PMSF must be added dropwise with vigorous st1rr1ng just prior to use. ' The number of strokes suggested is meant as a guide. Monitonng of cell lysis by microscopy is essential. • The suspension should become only slightly v1scous. If high·salt buffer is added too fast. DNA will be released. makmg the extract too viscous for homogenization and centnfugat1on

2.4 Formation of polyadenylation complexes 2.4.1 Introduction The endonucleolytic cleavage at the sile of future poly(A) addition only occurs after the substrate RNA has been assembled into a large complex that contains some or all the trans-acting processing factors. The composition of this complex is not known precisely since its formation has so far been analysed only in crude extracts. It is, however, quite clear that binding of CPSF to the AAUAAA sequence is the key event for complex formation (9). One of the cleavage factors may interact with a downstream sequence element and stabilize the CPSF-AAUAAA interaction (10). It is not known whether any of the other factors is stably associated \\ith the processing complex. A variety of experimental approaches can be made to investigate polyadenylation complexes:

(a) The formation of processing complexes may be detected by either glycerol gradient sedimentation {II) or, more conveniently, non-denaturing gel electrophoresis (see Section 2.4.2). (b) RNA can be extracted from the complexes. Analysis of this RNA by denaturing gel electrophoresis will reveal whether a complex contains substrates, intermediates, o r products of the reaction. (c) The region of an RNA substrate associated with processing factors can be investigated by nuclease digestion or chemical probing techniques (see Sections 2.4.3 and 2.4.4). 6

Elmar \Vahle and Walter Keller (d) Polyadenylation complexes separated by nati\e gel electrophoresis may be anal}sed with respect to their protein content by subsequent SDS polyacrylamide gel electrophoresis. UV cross-linking to the substrate RNA can identify those proteins that are in close contact with the R1 A (9). (e) The presence or absence of particular antigens or nucleic acids (e.g. snRNAs) in the polyadenylation complexes can be tested with blotting techniques (12).

2.4.2 Basic method Protocol2 describes the formation of processing (or polyadenylation) complexes using the nuclear extract prepared in Protocol 1. Protocol 2 forms the basis for many of the subsequent procedures. With slight modifications, it can also be used to analyse the binding of purified CPSF to AAUAAA-containing RNAs (see Section 3.2.5) or other RNA-protein interactions. Tbe polyadenylation complexes generated in Protocol 2 are visualized by nondenaturing (native) polyacrylamide gel electrophoresis. Complex formation requires ATP and so creatine phosphate is included in the reaction to replenish the ATP pool that is depleted b} ATPases in the nuclear extract. The extract contains adequate amounts of creatine kinase. Polyvinyl alcohol (PVA) is included in the reaction because it stimulates many macromolecular interactions, probably by increasing the effective concentrations of the reacting molecules ('excluded volume effect'). After the polyadenylation complexes have formed, heparin is added. Heparin is an anionic polymer that binds many nucleic acid-binding proteins and here serves to remove non-specific RNA-binding proteins from the precursor RNA. Protocol 2. Formation of polyadenylation complexes and their analysis by electrophoresis in native gels Equipment and reagents • Equipment for polyacrylamide gel electrophoresis

e 10mM MgCI7

• Dialysed Hela cell nuclear extract (see Prococo/ 11

e 12 5 % polyv1nyl alcohol IPVA. low molecular we•ght cold water soluble; S•gma)

•

e 2. 5 mg lml tRNA

32 P-Iabelled precursor RNA (approximately 500-1000 c.p.m fmoll. synthesrzed and capped tn vtrro (see Volume I. Chapter 1, Secuon 2.2)

• Hepaun 125 mg ml Sigma. grade I) • Loadmg buffer (10% glycerol 0.05% xylene cyanol, 0 .05% bromophenol blue)

• 0. 2 M OTT 1n 10 mM sodium acetate. pH 4 .8 . store at - 20 ° C

• Native gel electrophoreSIS buffer (25 mM Tris base, 25 mM borrc ac1d, 1 mM EDTA)•

• 10 mM A TP Dissolve A TP 1n water at approximately 0 1 M . Neutralize With ammoma, check1ng the pH with Indicator paper Determine the exact concentration by measuring the A 259 1~ 259 = 15 400). Store this stock solution at - 20 ° C. Dilute aliquots to 10 mM as required and store them also at - 20 ° C

• Acrylamide stock solution (20% acrylamide, 0 .25% bisacrylam1de in native gel electrophoreSIS buffer) • 10% ammonium persulphate (freshly made)

• 0 . 2 M creatine phosphate

eTEMED

7

Continued

1: 3' end-processing

of mRNA

Protocol 2 . Continued Method A. Preparation of the non-denaturing polyacrylamide gel

1. Clean glass electrophoresis plates, spacers, and, most of all, the comb thoroughly. 2. Assemble the glass plates (20 em long) with 1 mm thick spacers.

J . Make up a 4% polyacrylamide solution in nattve gel electrophoresis buffer tn a sufficient volume for the gel apparatus being used. For each 100ml mix: • acrylamide stock solution enative gel electrophoresis buffer

20ml 80ml

0.1 ml e TEMED 0.75ml • 1 0% ammonium persulphate 4. Pour the acrylamide solution between the glass plates, insert a well-forming comb, and leave the g•el to polymerize.

B. Preparation of the polyadenylation complexes 1. Prepare a reaction mtxture suffictent to enable 12 x 25 1-11 timed samples to be taken. To do this, dispense 120 fmol of RNA precursor (equivalent to 10 ng of an RNA 2Ei0 nt long) tnto a mtcrocentnfuge tube. Calculate the volume of RNA solution to be used from its concentration determined as described tn Section 2. 2. 2 . If the RNA has been stored as an ethanol precipitate, centrifuge it for 15 min in a microcentrifuge at 4 °C. Remove the supernatant as completely as possible since it contains the urea from the gel that was used to purify the RNA (see Volume I. Chapter 1, Section 2.3.3). Add 1 ml of 70% ethanol to the RNA pellet. centnfuge for 5 mtn, discard the supernatant and dry the pellet. Use a hand-held Ge1ger counter to control the recovery of radtoacttve RNA tn this and all subsequent steps. Proceed to step 4. 3 . If the RNA has already been precipitated and stored tn water or 50% ethanol, lyophtltze the appropnate quanttty 4. To dissolve the RNA pE!IIet, add the followtng reagents in the order gtven . • 2.5mg ml tRNA • H2 0 .o.2M

5

on

121-11 61-11 31-11

•10mM ATP

241JI

• 0.2 M creatine phosphate

30 1-11

• 10 mM MgCI 2

1 5 1-11

Vortex to mix and dissolve the RNA. Place the tube on ice and add 60 IJI of 1 2.5% PVA and 1 50 1-11 of nuclear extract • Mix gently by ptpetttng

6 . Wtthdraw a 251-11 aliquot into a fresh microcentrifuge tube and freeze it on dry ice.

Continued 8

Elmar \Vahle and Walter Keller Protocol 2. Contmued

7 . Transfer the rest of the reaction mixture to a 30 °C water bath. Take additional 25 ~I aliquots after 1, 2, 5, 10. 20, 30, 40, 50, 60, and 120 min. Freeze each on dry ice.

8 . At the end of the incubation, take all the tubes from the dry ice. Add 5 ~I of 25 mg/ml heparin to each and leave them for 10 min at room temperature.

C. Electrophoresis of polyadenylation complexes 1. Remove the comb from the non-denaturing polyacrylamide gel, assemble the apparatus and rinse the wells w1th native gel electrophoreSIS buffer.

2 . Prerun the gel for at least 1 h at 250 V . 3 . Load aliquots of all the samples into the wells of the gel. Use aliquots compatible with the size of the wells (5-30 ~1). 4 . Mix an aliquot of substrate RNA, that has not been incubated with proteins, with loading buffer. Load this sample into an empty lane of the gel•. 5. Run the gel at 2 50 V. The running time depends on the length of the precursor RNA; for an RNA of 250 nt, run the xylene cyanol marker dye in the lane containing free substrate RNA to the bottom of the gel 6 . Remove the gel from the electrophoresiS apparatus and separate the glass plates. 7 . With the gel on one of the plates, wrap 1t 10 plast1c cling film (e.g. Saran wrap). Place an X-ray film 1n contact with the gel and use a marking pen to mark the orientation of the gel on the film . 8. Expose the film at - 70 °C for an appropriate time and then develop 1t according to the manufacturers instructions. • Note that this buffer IS different from the TBE buffer normally used for gel electro· phoresis. ~ Less nuclear extract may be used. If so. add buffer D Isee Protocol 1) so that the combined volume of nuclear extract and buffer D is one half of the total reaction volume. ' loadmg buffer IS added only to the free substrate ANA but not to the aliquots from the reaction. These con tam suff1c1ent glycerol for loadmg .

The experiment shown in Figure /A demonstrates the time course of polyadenylation complex formation. The fastest migrating complex (complex H) represents the non-specific binding of proteins to the RNA. [ts formation does not require any of the polyadenylation signals. A second complex (S), the specific PQiyadenylauon complex, forms only ""ith RNAs that contain aJlthe sequences necessary for 3 ' end-processing. In the presence of ATP, this complex forms rapidly even at 0 °C. RNA that was at first bound by non-specific components (complex H) is also incorporated into complex S. Later, a third complex (P} appears that contains the cleaved and polyadenylated RNA product (see Section 2.5.2}. 9

1: 3' end-processing

of mRNA

A ..... X

z

I 0 I

2 5 '0 20 )() phata>e

,T·,z,

"""':~G:: ...

SnaJ.e 'cnom p} ropho>phatase

pXm 1 pYm I pZ + Pi

Note that initial cleavage with RNase T2 retains the methylated nucleoside' (Xm, ym) and another residue (Z) in phosphodiester linkage as part of a

extende-d cap structure. By contrast. nuclease PI treatment releases the ca, and y m and Z as 5' -mononucleotides; nuclease PI also has a 3' -phosphatase activity. The constituent nucleotides in the capped oligonucleotide can be identified at various stages b:y two-dimensional thin-layer cellulos~ chromatography (43).

4.2 E.nzymic analysis of caps 4.2 .1 Removal of caps from mRNA Protocol 9 describes the removal of caps from mR As asing either nuclease: PI or J~Nase T2. This procedure may also be employed for preparing radiolabelled cap structures for use as markers for analytical purposes, e.g. see Figures 5 and 6 (Section 4.3). 54

Yasuhiro Furuichi and Aaron /. Shotkin Protocol 9 . Removal of caps with nuclease P1 or RNase T2 Reagents • Aad1olabelled capped mANA (- 1-2 mgl ml) prepared as m Protocols 1-4.

• ANase T2 !Calb1ochem) • 0.1 M sod1um acetate buffer. pH 6 0

6 -8

e 0.1 M sod1um acetate buffer, pH 4 . 5, contam1ng 10 mM EDTA

• Nuclease P1 !Calb10cheml

Method A. D1gestion with nuclease P1 1. Dilute the capped RNA to a final concentration of -0.5 mg/ml and 10 mM sodium acetate buffer. pH 6.0. using the 0.1 M stock solution of 0.1 M sodium acetate, pH 6.0 . 2. Add nuclease P 1 to 50 JJg/ml. 3. Incubate the mixture at 37 °C for 30 min. 4 . Proceed to Protocol 16 (or another purification or analytical technique)

B. Digestion With RNase T2 1. Dilute the capped mANA to a fmal concentration of - 0. 5 mg ml in 0 1 ml of 10 mM sodium acetate, pH 4. 5, and 1 mM EDTA using the 0.1 M stock solution of 0 . 1 M sod1um acetate, pH 4. 5, 10 mM EDTA. 2 . Add RNase T2 to 50 U ml final concentration. 3 . Incubate the mixture at 37 °C for 2 h. 4 . Proceed to Protocol 12 (or another purification or analytical method).

4.2.2 Enzymatic cleavage of caps with nucleotide pyrophosph atases Protocols 10 and II described the cleavage of caps with snake venom pyro-

phosphatase and tobacco pyrophosphatase, respectively. Protocol 10 . Cleavage of caps with snake venom pyrophosphatase to identify the Nm in m 7GpppNm Reagents • m 7GpppN'" caps removed from capped mANA by nuclease P1 digest1on !see Protocol 91 and isolated by paper electro· phores1s as 1n Protocol 14 (Secuon 4.3 21. see also Figure 68

• Snake venom nucleotide pyrophosphatase (Sigma) dissolved m H 20 at - 1 Ul ml e 1 0 M Tris-HCI, pH 7 5 e 10 mM MgCI,

Method 1. Dissolve the caps 1n 8 JJI of 20 mM Tris-HCI, pH 7. 5, and add 1 JJ) of 10 mM MgC12 and 1 JJI of nucleotide pyrophosphatase

Continued 55

2: Capping and methylation of mRNA Protocol 10 . Continued 2. Incubate the mixture at 37 °C for 30 min. 3. Freeze and lyophilize the sample for subsequent analysis by high-voltage paper electrophoresis (see Protocol 16) or two-dimensional chromatography (see Protocol 7 7 and Figure 7).

Treatment of capped mRNA with tobacco pyrophosphatase is used to prepare uncapped mRNA (containing a 5 '-terminal pNm) by releasing the cap constituents as m7Gp and Pi. After removal of the 5 '-monophosphate by phosphatase treatment, the RNA can then be 5 ' end-labelled by incubation with polynucleotide kinase and [ y-32 P] ATP (Volume I, Chapter 2, Protocol 2). Protocol 1 1. Removal of caps from m ANA with tobacco pyrophosphatase

Equipment and reagents one of the methods described tn Volume I, Chapter 1, Section 3

• 0.25 M sodtum acetate buffer, pH 6.0

e o. 125M on • Tobacco actd pyrophosphatase (Sigma, -1000U/mll • Capped mRNA (1 mgl mll isolated by

• Phenol:chloroform, elution buffer, Sephadex G-1 00 column, and 3 M potassium acetate, pH 4.5, as described in Protocol 1

Method 1. Mix in a microcentrifuge tube: • 0.25 M sodium acetate buffer

1 (JI

e 0.125M OTT

11JI

• tobacco acid pyrophosphatase

1 (JI

• capped mANA

1 (JI

• H2 0

to 10 (JI final volume

2. Incubate the mixture at 37 °C for 1 h. Stop the reaction by placing the tube on ice. 3 . Spot an aliquot of the reaction mixture on paper and analyse it according to Protocol 1 7, (Section 4 . 3. 5; see also Figure 7) for confirmation of the release of m 7 Gp (identified by scintillation counting if radiolabelled or by fluorescence under UV light if present in sufficient amount).

4. Purify the decapped RNA according to Protocol 1, steps 3 - 8.

The decapped RNA can be 32P-Iabelled at the 5' -end by first removing the residual phosphate by phosphatase treatment (e.g. with I0 units of calf intestinal alkaline phosphatase per millilitre in 50 mM Tris-HCI, pH 8.0, for 60 min at 37 °C} and then incubating the repurified RNA with polynucleotide kinase and [y-32P]ATP (44). 56

Yasuhiro Furuichi and Aaron ]. Shatkin

4 .3 Analysis of caps following enzymic digestion 4.3.1 Chromatography on DBAE- cellulose and DEAE- cellulose Dihydroxyboryl aminoethyl cellulose (DBAE) is a derivative of DEAE-cellulose containing covalently-bound dihydroxyboryl groups. Its use as an affinity matrix depends upon the selective affinity of 2 1 -3 1 cis-diols for dihydroboryl groups. DBAE-cellulose was developed originally for the isolation of 3 1 -terminal oligonucleotides from high molecular weight RNA (45). However, it can also be used to isolate the 5 ' -terminal capped oligonucleotide from RNA digests since the cap possesses a 2 ' -3 1 cis-d.iol on the ribose moiety of m7 G. The procedure is described in Protocol 12. Caps removed from mRNA by enzymic digestion (Section 3.2) may also be separated by chromatography on DEAE-cellulose. Protocol 13 describes the initial separation of caps, which elute from DEAE-cellulose between 0.2 M and 0.25 M NaCI, and subsequent concentration and desalting of the caps. Protocol 12. DBAE-cellulose chromatography Equipment and reagents • Application buffer (1.0M NaCI, 0 . 1 M MgCI2, 20% DMSO, 50 mM morpholine. pH 8.71

• Capped mRNA digested with RNase T2 as described in Protocol 9 ; this will be -0.5 mg/ml in 10 mM sodium acetate, pH 4.5, 1 mM EDTA

• Application buffer plus 1.0 M sorbitol (J . T . Baker)

e o.5 M morpholine buffer, pH 8.7 • 5.0 M NaCI

e DBAE- cellulose column (15 x 0.6cm) equilibrated in application buffer

• 1.0 M MgCI 2 • Dimethylsulphoxide (DMSO; Pierce HPLC, spectrograde)

Method 1 . Adjust the composition of the RNase T2-treated starting sample to that of application buffer (buffer A) as follows :

• RNase T2-digested capped mRNA

250 ~I

• 0. 5 M morpholine, pH 8 . 7

100 ~I

• 5.0 M NaCI e 1.0M MgCI 2

200 111

• DMSO

200~1

100 ~I

• H2 0 to 1 ml final volume 2 . Apply the sample to the equilibrated DBAE- cellulose column. 3 . Develop the column with application buffer at a flow rate of - 5 ml/h and collect 1 ml fractions.

Continued

57

2: Capping and methylation of mRNA Protocol 12. Continued 4. Assay -

25~-JI

aliquots of each fraction by scintillation counting.

5 . After most of the 3'-nucleotides (resulting from the digested mRNA) have eluted from the column, change the elution abruptly to application buffer plus 1.0 M sorbitol and continue the elution at a flow rate of -6-7 ml/h. 6 . Combine the fractions containing the caps (they elute within - 1 0 ml after changing to the sorbitol-containing buffer) and proceed to the next procedure, e.g. DEAE-cellulose chromatography as described in

Protocol 13.

Protocol 13. DEAE-cellulose chromatography

Equipment and reagents • Caps isolated by DBAE-cellulose chromatography (sorbitol-eluted fractions from Protocol 12, step 61

and 1.0 x 0.6 em) equilibrated with 20 mM Tris- HCI, pH 8.0, containing 7 M urea • Yeast tRNA digested w1th pancreatic RNase; incubate 1 mg/ml tRNA• with 0. 1 mg/ml of pancreatiC RNase for 6 h at 37 ° C m 0.1 M Tris-HCI. pH 8 .0

e 1.0 M Tris-HCI, pH 8.0 e 50 mM NaCI in 20 mM Tns-HCI, pH 8.0. containing 7.0 M urea e 0 . 3 M NaCI in 20 mM Tris-HCI, pH 8.0, containing 7.0 M urea • DEAE-cellulose columns (40 x 0 .6 em

• A senes of freshly-prepared ammonium bicarbonate solutions (50 mM, 75 mM. 0 .1Mand1.0M)

Method 1. Dilute the combined fractions of the sorbitol-eluted caps 10 -fold with

H2 0. 2. Add, as a marker for the oligonucleotide elution posit ion, 0 . 5 ml (- 20 A 260 units) of a pancreatic RNase digest of yeast tRNA. 3 . Apply the mixture to the DEAE-cellulose column and wash the sample in 20 mM Tris - HCI, pH 8 .0 , containing 7 M urea. 4 . Elute the column with a linear salt gradient of NaCI (50 ml each of 50 mM and 0.3 M NaCI in 20 mM Tris-HCJ. contai ning 7 M urea). 5 . Monitor the eluate continuously at A 260 (or by scintillation counting if radiolabelled yeast tRNA has been used) for mononucleotides (- 2 charge). dinucleotides (- 3 charge ). etc. 6 . Collect the fract1ons containing caps (position of -4 to -6 charge, depending on extent of 2'-0-methylation). 7 . Desalt and concentrate the samples containing the caps by DEAE chromatography by diluting the combined fractions 10-fold with H20 and applying the pooled sample to a small DEAE column (0.6 x 1 em).

Continued 58

Yasuhiro Furuichi and Aaron ]. Shatk:in Protocol 13. Continued 8. Wash the column successively with 5 ml each of the freshly prepared 50 mM , 75 mM, and 0.1 M NH 4HC03 solutions. 9 . Finally elute the caps with 1 ml of 1 M NH 4 HC 0

3

and lyophilize the eluate.

• Radiolabelled tRNA may be used.

Figure 5 illustrates the combined use of DBAE-cellulose and DEAE-cellulose chromatography. In this example, 32P-labelled avian sarcoma virus RNA (ASV; 1.1 x 107 c. p.m., specific radioactivity 1.5-3.0 X 107 c.p.m ./~g) was isolated from purified virus (46), mixed with [3H] methyl-labelled reovirus mRNA synthesized in vitro (see Protocol I) (I 0 ~g. I x lOS c. p.m.) and digested with 50 units of RNase T2 (see Protocol 9). After incubation for 6 h at 37 °C, the mixture was applied to a DBAE-cellulose column (see Protocol 12). After the decapped 32 PIabelled ASV RNA had been removed by washing the column with application buffer, the column was eluted by applying buffer containing I M sorbitol (see Figure 5A) as described in Protocol 12, step 5. The 32P-Iabelled caps eluted in the same fractions as the [ 3H]methyl-labelled reovirus mRNA caps, immediately after the buffer change. The combined fractions containing the caps were then analysed by DEAE-cellulose chromatography (Figure 58) as described in Prorocol 13. A major peak of radioactivity with a net charge of about - 4.5 , which corresponds to the Cap 1 structure (m7GpppGmpCp), was resolved by this procedure. A minor peak eluted later with a net negative charge of about - 5.5; it corresponds to Cap 2 structures containing the ring-opened derivative of m7G (m7'G) which has lost the+ charge (46). These values are based on theoretical charges of + I at the N7 position of m7G, -3 for GpppNm, and approximately - 3 and -4 for pNp and pNmpNp, respectively. As shown in Figure 68 (inset), m7GpppGm elutes from DEAE-cellulose with a net charge between -2 and - 3, based on the elution of nucleotide markers. The divergence from the theoretical charge of - 2 ( + 1 - I - I - I) may be due to the stacking effect of the nucleosides in m7 GpppGm, resulting in stronger binding to the DEAE-cellulose matrix.

4.3.2 Chromatography on paper Cap structures and their derivatives can be resolved by descending paper chromatography on Whatmann 3MM paper in isobutyric acid: 0.5 M NH 40H 00:6 v/ v) as described in Prorocol 14. The R F values of caps and their derivatives in this system are: m7GpppAm, 0.40; m7 Gpppom, 0.36; GpppA, 0.25; GpppC, 0.16; GpppG, 0.08; and GpppU, 0.08. It is important to note that m 7G residue of the cap is sensitive to alkaline conditions and is degraded to products that include the 'ring-opened' form in which the 8-9 bond of the m7G imidazole ring is cleaved with an accompanying loss of the positive charge. Therefore, it is not appropriate to use alkaline solvents for the characterization of caps. 59

2: Copping and methylation

of mRNA

A 24

I

I

1

r8ufltr A

20-

..- 16 I

0

~

~

)(

12~

Bulftr A • Sor!Htol

~

~

RNase T2 digested ASV RNA

.........-

Caps

E ~ a.

4-

\

0

I

20

0

40

60

10-

-

8

-

6 E

0 ~

)(

.

0.

-

c. sr--

..

-

-

4 0 :::

2

eh

Fraction No.

B

..

..

I 0

I 0

~

~

)(

>
.1racL~ from T. brucei (71 ). The relationship of the total cell RNA ligase acthity to the mitochondrial activity described in L. tarentolae is not clear. The L. rarentolae RNA ligase acti\ ity co-sediment~ with the kinetoplast-mitochondnon fraction (50). It is solubilized in Triton X-100 and thus can be assayed in mitochondrial extracts. Incubation of cytosolic RNA with mitochondrial TL extract and [ ~- 32 P) UTP under the conditions required for TUTase in the presence of ATP and Mg2 + ions yields a 180 nt labelled RNA product whose relative electrophoretic mobility varies with the gel concentration, a behaviour characteristic of circular RNA molecules. RNA ligase is quantitatively assayed by the addition of [ a- 32 P) pCp to the 3 ' termini of RNA molecules as described m Protocol 13. 99

3: RNA editing in mitochondria Prot ocol 13 . Assay of RNA ligase in mitochondrial extracts

Reagents e Mitochondrial extract see Protocol 11 I

m . TS, or S-1 00;

3000Ci.m tmol, 10mCi mil

e Cytosolic RNA substrate 1500 ~Jglml ) prepared as described in Protocol 12 • 10mM ATP

• 1 .OM MgCI2 e 0 . 25 M Hepes- KOH, pH 7 .9 • 0 . 1 M OTT

• ( a · 32P I pCp (New England Nuclear;

e OMSO • RNAguard IPharmacia, # 27-0815·0 1 331Ji ml) • DE81 discs and 0 .5 M sodium phosphate buffer, pH 6.8, wtth 0 . 5% sodtum pyro phosphate (see Protocol 9 1

Method 1. In a microcentrifuge tube on ice, m1x: • cytosolic RNA substrate

1-51JI

e 1 .0 M MgCI 2 e 0 . 1 M OTT

2 Ill 3.31JI

e 0 .25 M Hepes-KOH, pH 7.9

20 Ill

• lOmMATP e OMSO

11JI 10111

• [a- 32P]pCp e RNAguard • mitochondrial extract • H20

2111 1 IJI 1 0 IJI to 50 Ill final volume

2. Incubate the mixture at 4 °C for 1 5 h. 3 . Measure the addition of [a-32P]pCp to the RNA substrate by spotting aliquots of the mixture on DE81 filter discs and processing them as described in Protocol 9 , steps 4-6.

8.5 Cryptic RNase A sequence- or structure-specific cryptic RNase activity can be detected in mitochondrial extracts (TL, TS, or S-1 00 extracts, see Protocol 11 ). This en ptic RNase can be activated either by the addition of heparin (5 )Jg/ ml) o by predigestion of the lysate with proteinase K. Protoco/14 describes the activation of the cryptic RNase and its subseq tent assay. The RNA substrate used for this assay is a 200 nt RNA synthesized b> in vitro transcription from a n bacteriophage promoter using T7 RNA polymerase. The template DNA consists of the 22 nt PER of the cytochrome b gene together with 56 nt of 5 ' flanking sequence, 186 nt of 3 ' flank ing sequence, and 73 nt of Bluescript vector sequence at the 5' end. Cleavage bY the cryptic RNase occurs at one major site and four minor sites within the P ER· 100

Larry Simpson. Agda M. Simpson, and Beat Blum Protocol 14. Assay for a sequence- or structure -specific cryptic RNase activity in mitochondrial extracts

Equipment and reagents Smal site of pBiuescript SK l- l vector from Stratagene)

• Mitochondnal extract (Tl, TS, or S- 100, see Protocol 77 1 • Hepann (S1gmal • Proteinase K tBRL): dissolve the prote1nase K at 10 mgfml in 50 mM Tns-HCI, pH 8.0. 1 mm CaCI 2 e 10 mM ATP e 10 x buffer {30 mM MgCI2, 0.1 M TrisHCI, pH 7. 5, 50 )Jgfml heparin) • pNB2 plasmid [a 198 bp Acci!Rsal restriction fragment from the plt120 maxicircle region containing the 5 '-end of cytochrome b gene cloned into the

e pNB2 RNA substrate (108 c.p.m 11Jgl: transcribe BamHI·digested pNB2 plasm1d DNA in vitro us1ng T7 RNA polymerase and (a- 32P]UTP as described 1n Volume I, Chapter 1 !Protocol4 ) or Chapter 2, thiS volume (Protocol 4 I • Phenol:chloroform ( 1: 1, vlvl and 2M NaCI as Protocol 3 • 8% polyacrylamide/7 M urea denaturing (sequencing) gel, loading and electrophoresis buffers and equipment (see Volume I, Chapter 1, Protocol 8 or Volume I. Chapter 4, Protocol 11 I

Method Activation of the cryptic RNase 1. Either add proteinase K (1 00 l)g/ml final concentration) to the mitochondrial extract and incubate the extract for 5 min at 37 °C or add heparin to 5 ~Jg /ml. Activation by protease predigestion and heparin are synergistic and together result in better cleavage.

2. In a microcentrifuge tube on ice, mix: 5 ~I 51)1

• 10 x buHer e lOmM ATP pNB2 RNA substrate ( 10 4 c.p.m.)

1 ~JI

• activated mitochondrial extract (from step 1)

10 ~I

• 32P-Iabelled

to 50~~ final volume

• H2 0 3. Incubate the mixture for 1 h at 27 °C.

4. Phenol extract the RNA and recover the RNA by ethanol precipitation as described in Protocol 3, steps 13- 16. 5 . Add gel loading buffer ( 10 IJI of 10M ureal and analyse the cleavage products by gel electrophoresis and autoradiography as described in Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4, Protocol 11.

8.6 gRNA:m.RNA chimaera-forming activity In order to study the first step of the proposed trans-esterification model for RNA editing (16, 17), the authors developed a cell-free system using an extract from mitochondria prepared by sonication followed by salt extraction. Chimaera formation is monitored by the covalent transfer of uniformly-labelled 32P-gRNA 101

3: RNA editing in mitochondria to a higher molecular weight non-radioactive test mRNA by gel electrophoresis. Both the 32 P-gRNA and the test mRNA are synthesized by T~ transcription from PCR-derived DNA templates (63). Sequence analysis of the reaction products by selective PCR amplification and cloning revealed gRNA:mRNA chimaeric molecules as the most prominent products (20). This in vitro system has been used to confirm the importance of the anchor sequence between the gRNA and mRNA, and should be useful for developing conditions for complete in vitro editing (20). The system is described in Protocol 15.

Protocol 15. gRNA:mRNA chimaera-forming activity

Equipment and reagents • Centricon 10 centrifugal centrators (Amicon 4205)

microcon-

• Kinetoplast-mitochondrion fraction isolated from one litre of L. tarentolae according to Protocol 6. Keep the kinetoplast-mitochondrion fraction at - 70 °C until extraction is performed 32

•Uniformly-labelled P-gRNA prepared by in vitro transcription with [ a-32P) UTP and T7 RNA polymerase (Volume I, Chapter 1, Protoco/4 or Chapter 2, this volume, Protocol 4) and isolated by gel electrophoresis (Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4,

Protocol 1 1) • Test mRNA containing the PER plus flanking (anchor sequence) region; prepare this by in vitro transcription with T7 RNA polymerase followed by gel electrophoresis (see above) e1.0 M Hepes-NaOH, pH 7.9 e 0. 5 M EDTA (adjust the pH to 8.0 w 1th NaOH)

e Q.1 M DTT e 0 .1 M PMSF (Sigma # P-7626) in iso· propanol • Human placental RNase Inhibitor IBRL #5518SA) • 20 mg/ml proteinase K (BRL # 5530UA ) stock • Hypotonic buffer (1 0 mM Hepes-NaOH, pH 7.9, 0.5 mM EDTA) • Extract buffer (20 mM Hepes-NaOH. pH 7.9, 20% glycerol, 0.1 M KCI, 0.2 mM EDTA, 0.5mM PMSF, 0 .5mM DTT prepared fresh from stock solutions) • 2 x salt extraction buffer (40 mM Hepes, pH 7.9, 50% glycerol. 0.84 M NaCI. 0.4 mM EDTA, 1 mM PMSF, 1 mM DTT. prepared fresh from stock solutions) • Annealing buffer (20 mM Hepes-NaOH. pH 7 .9 , 0 . 1 M KCI, 1 mM EDTA) • Reaction buffer (8% PEG 8000, 12.5 mM MgCI 2 , 2. 5 mM A TP. 1 unitl~l RNase inhibitor) • Proteinase K solution (0.25% N-lauryl sarcosine, 25 mM EDTA , 0.25 mglml proteinase K)

• Glycerol (ultrapure; BRL 5514UA) e2.0 M NaCI e1 .0 M KCI

• Sonicator (Braunsomc microtip; Braun)

e0. 1 M MgCI2 •13% (wl v) polyethylene glycol 8000 (PEG 8000; Sigma #P-2139) • N-laurylsarcosine (Sigma # L-51 25)

# 451 0

w ith

• Materials and equipment for poly acrylamidel urea gel electrophoresiS as described in Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4 .

• 20 mM ATP (Pharmacia # 27-2056-01)

Protocol 11

Continued 102

Larry Simpson. Agda M. Simpson, and Beat Blum

Protocol 15. Continued Method 1 . Resuspend the kinetoplast-mitochondrion fraction from one litre of cell culture in 2 ml of 1ce-cold hypotonic buffer. Keep this on ice for 10 min to allow the mitochondna to swell. 2 . Disrupt the swollen mitochondria by sonication at 5 °C using three 20 sec periods at 100 watts. 3 . Immediately add 2 ml of 2 x salt extraction buffer and gently agitate the solution with a small magnetic stirrer for 30 min on ice.

4. Clarify the extract by centrifuging at 50 000 g for 30 min at 4 °C. 5 . Concentrate the extract at 4 °C on two Centricon 10 microconcentrators to an approximate vel. of 100 IJI each.

6 . Change the buffer at 4 °C using the same microconcentrators by applying two 2 ml lots of extract buffer to each. 7. Quick freeze 200 IJI aliquots of the pooled extracts in dry ice/ethanol and store them at - 70 °C. Chimaera-forming activity is maintained for up to 3 months. 32P-Iabelled gRNA and the test mANA in 2.51JI of annealing buffer. Denature the RNA for 3 min at 70 °C then anneal them at 37 °C and 25 °C for 10 min each.

8 . Anneal equimolar amounts of the

9. Add 81JI of reaction buffer and 1 5 IJI of thawed mitochondrial extract {from step 7) to the annealed RNAs. Incubate for 15-120 min at 27 °C.

10. Stop the reaction with 100 IJI of proteinase K solution and incubate at 37 °C for 20 min.

11 . Recover the RNA by phenol extraction and ethanol precipitation as described in Protocol 3, steps 13- 16.

12. Analyse the resulting RNA chimaeric products on denaturing acrylamide/ urea gels followed by autoradiography {Volume I. Chapter 1, Protocol 8, or Volume I, Chapter 4 , Protocol 11 ).

The RNA products from Protocol 15 may also be analysed by sequence determination following RT-PCR and cloning. In order to amplify specifically the exogenous RNA, a 'tag' sequence may be added to the test RNA. Further details of RT -PCR can be found in Volume I, Chapter 2, Section 3.4 and Volume I, Chapter 3, Section 2.2.3

Acknowledgement This work was supported in part by NIH research grant Al09102.

References I. Hodges, P. E., Navaratnam, N., Greeve, J. C. and Scott, J . (1991). Nucleic Acids Res., 19, 1197. 103

3: RNA t>diting in mitochondria 2. Lau, P. P, Xiong.\\ . Zhu, H.-J ., Chen, S -H .• and Chan, L. (1991) J. B IJ/, Chem., 266, 20 550. 3. Gualberto, J. M., Lamattina, L., Bonnard, G., Wei!, J . H., and Greinenberger, J M. (1989). 1\'ature, 341, 660. 4. Covello, P . S. and Gray, M. W . (1989). 'Vuwre. 341, 662. 5. 'vtahendran, R., Spothwood, '.1. R, and \Iiller. D L. (1991) \'ature, 349, 43.;. 6. Simpson L. and Shav., J. (1989). Cell, 57, 355 7. Vidal, S., Curran, J., and Kolakofsky, D . (1990). EMBO J. , 9. 2017. 8. Driscoll, D., Wynne, J ., Wallis, S., and Scou, J. (1989). Cell, 58, 519. 9. Chen, S.-l·l., Li, X, Liao, \\' . S. L., Wu, J . H .. and Chan L. (1990) J. Btol. Chem .. 265, 6811 10. Lau, P. P., Chen, S.-H., Wang, J. C., and Chan, L. (1990). Nucl. Acids Res 18, 5817. II. Smith. H. C .. Kuo, S.-R .. Backus. J. W . Harris, S. G., Spark , C. E., and Sparks, J D. (1991) Proc. /'vat/ Acad. Sci. USA, 88, 1489. 12. Greeve, J., Navaratnam, .. and Scott, J. (1991) . •Vue/, Acids Res. , 19, 3569 13. Backus, J. W. and Smith, H . C. (1991). Vue/. Acids Res.. 19, 6781. 14. Simpson, L. (1990). Science, 250, 512. 15. Blum, B., Bakalara, N., and Simpson, L. (1990} Cell, 60, 189. 16. Blum, B, Sturm,, . R., Simpson, A. \1 .. and Simpson L (1991). Cell. 65 3. 1'. Cech, T R. (1991). Cell, 64, 667. 18. Koslowsky, D J, Gringer, H . U., Morales, T. H., and Stuart, K. (1992). Nature. 356, 807 19. Harris, M. E. and HaJduk, S. L. (1992}. Cell, 68, 1091 .:!0. Blum. B. and Simpson, L. (1992). Proc. \'all A cad. Set L.SA. 89. II 9~ 21. Simpson, L (1987). Annu. Rei'. .\1tcrobtol.• 41, 363. 22. Sturm, N. R. and Simpson, L. (1991). Nucl. Actds Res., 19, 6277. 23. Pollard, V. W., Rohrer, S. P., Michelotti, E. F., Hancock, K., and Hajduk, S L. {1990). Cell, 63, 783. 24. Kidane. G, Hughes, D, and Simp~on, L {1984) Gene, 27. 265. 25. Maslo", D. A. and Simpson, L. (1992). Cell, 10, 459 26. Morel, C., Chiari, E., Camargo, E., Mattei, D., Romanha, A . and Simpson, L. (1980). Proc. Nat/ Acad. Set. USA, 77, 6810. 27. Sturm. "1. R. , Degra\e, W., Morel, C .. and Simpson, L (1989). Mol Bwc rem. Parasitol., 33. 205. 28. Gomez-Eichelmann. M. C., Holz, G., Jr , Beach, D., Simpson. A. M .. and Simpson, L. (1988). Mol. Biochem. Parasitol., 27, 143. 29. Simpson, L. (1972). Int. Rev. Cytol., 32, 139. 30. Stuart, h. 0991). A111111. Re1·. .\llcrobiol. 45 327 31. Benne, R (1989). Biochim. Bwphys. Acta, 1007, 131. 32. Feagin, J and Stuart. K. (1988). ~to/. Cell Bioi., 8, 1.259 33. Feagin, J., Jasmer, D., and Stuart, K. (1987). Cell, 49, 337 34. Duszenko, M., Ferguson, M ., Lamont, G., Rifkm. M., and Cros'i. G. (1985). J. £r:p. \1ed., 162, 1256. 35. Briones, \.1. R. S., Nelson, K., Beverle}. S. M , .\ffonso. H. T. Camargo. E. P .. and Floeter-Winter, L. M. (1992}. 'viol. Biochem. Parosilol., 53, 121 . 36. Simpson, L. and Berliner, J . (1974). J. Protozoal., 21, 382. 37. Simpson, L. and Simpson, A (1974) J Proto::.ool., 21. 7i'4.

104

Lorry Simpson, Agdo M. Simpson, and Beat Blum 38. Simpson, L (1979). Proc '\'at/ Acad. Sci. USA, 16, 1585. 39. Ryan, K A., Shapiro, T. A., Rauch, C. A., and Englund, P T (1988). Annu. Re~ . Microbtol., 42, 339. 40. Hajduk, S., Klein, V., and Englund, P. {1984). Cell, 36, 483. 41. Tibayrenc, M., \\ard, P ., Moya, A ., and Ayala, F. (1986). Proc . .Vat/ Acad Set. USA, 83, 115 . 42. Brunk, C. and Simpson, L. (1977). Anal. Biochem. , 82, 455 43. Goncalves, A . M., Nehme, N. S., and Morel, C. M. (1984). In Genes and antigens of paraslles (ed. C. M. Morel), p. 95. Fundacao Oswaldo Cruz, Rio de Janeiro. 44. Beidler, J. L., Hilhard, P. R., and Rill, R. L. (1982). Anal. Biochem, 126, 374 ~5. Lopes, U., Momen, H., GrimaldJ, G., 1\larzocht, M., Pacheco, R , and Morel, C. (1984). J. Parasitol., 70, 89. 46. Morel, C. and Simpson, L. {1980). Am. J. Trop. Med. Hyg., 29, 1070. 47. Avila, H., Goncalves, A.M., Nehme, N. S., Morel, C. M ., and Simpson, L. (1990). Mol. Biochem. Parasito/., 42, 175. 48. Simpson, L. {1968). J. Proto:.oo/., 15, 132. 49. Braly, P ., Simpson, L., and Kretzer, F (1974) J. Proro;:ool, 21, 782. 50. Bakalara, N., Simpson, A.M., and Simpson, L. (1989). J. BIOI. Chem., 264, 18 679. 51. Simpson, L. and Simpson. A. (1978). Cell, 14, 169. 52. Simpson, A. \1., Suyama, Y , De\~e~. H., Campbell, D. and Simpson, L. (1989) Nucl. rlctds Res. , 17, 542.,. 53. Hancock, K. and Hajduk, S. L. (1990). J. Bioi. Chem., 265, 19 208. 54. Maslov, D. A., Sturm, N. R. • Niner, B. M., Gruszynski, E. S., Peris, M., and Simpson, L. ( 1992). ~1o/. Cell. Bioi., 12. 56. 55. Sturm, . R. and Simpson, L. (1990). Cell, 61, 8"1. 56. Sha\\ , J , Campbell, D., and Simpson, L. (1989). Proc. Sat Acad. Set., 86,6220. 57. Blum , B. and Simpson, L. (1990). Cell. 62, 391. 58. Masuda, H .. Simpson, L., Rosenblatt. H., and Simp~on, /\. (1979). Gene, 6, 51. 59. \\ood, W, Gitschier, J ., La5k)', L., and La\""· R. (1985). Proc. Nat/ Acad Sci USA, 82, 1585. 60. Abraham, J., Feagin, J., and Stuart, K. (1988). Cell, 55, 267. 61. Decker, C. J. and Sollner-Webb, B. (1990). Cell, 61, 1001. 62. McPherson, M. J., Qutrke, P., and Taylor. G. R. (ed) !1991). PCR: a practical approach. IRL Press, Oxford. 63. 1\.·f llljgan, 1. F., Groebe, D. R., Witherell. G. W., and Uhlenbecl.:. 0. C. (1987). Nuc/. Actds Res., IS, 8783. 64. Von Haeseler, A .. Blum, B , Simpson, L .. Sturm,'-· and \\aterman. M.S. (1992). 'Yuc/. Actds Res.. 20, 2717. 65. Sha\\. J , Feagm, J . E., Stuart, K .. and Simpson, L. (1988) Cell, 53, ~01 66. \an der Spek. H , ArtS, G.-J ., ZwaaJ, R. R., Van den Burg, J . , Sloof, P ., and Benne, R. (1991). EMBO J., 10, 1217. 67. Simpson, A M , Bakalara, N .. and Simpson, L. (1992). J Btol Chem., 267,6782 68. KoslO\\Sky, D J., Bhat, G J .. Read, L. t.., and Stuart, K (1992). Cell, 61, 5r 69. Rc:ad, L. K., Myler, P. J ., and Stuart, K. (1992). 1. B10/. Chem, 267, 1123. 70. Read, L. K., Corell, R. A., and Stuart, K. (1992). Nuc/. Acids Res., 20, 2341. 71. White, T. and Bor~t. P. ( 1987). Nuc/. AC'ids Res. , IS, 3275.

105

Analysis of messe11ger RNA turnover in cell-free extracts from mammalian cells JEFF ROSS

1. Introduction Messenger RNA turnover innuences gene expression in most or all cells, from bacteria to mammals (I, 2). Two sorts of observations support this notio n . First, the steady-state levels of many mRNAs are determined more by their half-lives than by the rates at which their genes are transcribed. Second, the half-lives of some mRNAs are not fixed but vary in response to the nutritional status of the cell, its position in the replication cycle, or its stage of differentiation. Many important questions need to be answered to understand how mRNA turnover is regulated and how it innuences cell growth, differentiation, and neoplastic transformation. How many mRNA-degrading enzymes exist per cell? Which are endonucleases and which exonucleases, and at what site in a mRNA molecule do they first begin the degradation process? What is the pathway of mRNA decay, i.e. what are the degradation intermediates? What sequences in a mRNA determine its half-life? How does translation affect mRNA turnover? What trans-acting factors regulate mRNA turnover and how do they function? These questions illustrate the need to use in vitro systems to investigate mRNA turnover, particularly in cells of higher organisms in which genetic analysis is difficult. In ~·itro systems offer several advantages (Table 1). Two of these are particularly imponant. (a) mRNA decay intermediates that are difficult or impossible to detect in intact cells can sometimes be identified in cell-free systems. The average intracellular half-life of an mRNA might be 20 h, but, once each individual mRNA molecule begins to be destroyed, the n:action is apparently complete in minutes or seconds. If degradation occurrc!d sJo,\ly, it would be eas) to detect mRNA decay intermediates in cells, which, with few exceptions, is not the case. Since degradation rates are slower in vitro than in cells, it is possible to identify intermediates.

4 : mRNA turnover in cell-free extracts Table 1. Advantages of analysing mANA stability in cell -free extracts • mANA decay intermediates which are difficult or impossible to observe in cells can be detected in VItro. • Messenger ribonucleases and trans-acting factors which stabilize mANA can ,e assayed and purif1ed • Relat1ve mANA turnover rates can be measured Without us1ng mh1b1to ·s of transcnpt1on. most or all of which are toxic to cells and not specific for transcnption. • The effects of mANA sequences and structures on half-life can be readily mvest1gated.

(b) In vitro mRNA decay systems, Like in vitro transcription systems, permn the investigator to detect and purify enzymes, co-factors, and trans-acung regulators. Even if practical schemes are available to select mutations m genes involved in mRNA turnover, some of the interesting mutants m1~ht be lethal, making it necessary to devise in vitro assays to characterize the relevant gene products. This chapter focuses on three in vitro methods currently used to investigate mRNA turnover in cells of higher organisms. It is intended as a guide and by no means presents all possible assay schemes. Improvements on the curre!l! systems \Hll surely continue to be made. The advantages and disad' amages of each system wiU need to be considered by the investigator in the context of the experiments. ln choosing a suitable in vitro system, it is important to remem ber that relative mRNA half-lives in vitro should re nect those in cells: mR NAs that are stable in cells should also be stable in vitro and, conversely, unstable mRNAs should be unstable in both. Half-Hves in cells and in vitro are unlikely to be identical, since mRNA decay occurs more slowly in vitro. However, if LWO mRNAs are degraded at rates differing by 20-fold in cells but by 3-fold or le)s in vitro, then the extract or reaction conditions should be modified. For example, mRNA-specific regulatory factors might have been inactivated during extract preparation. The protocols in this chapter suggest some options for modifymg each system.

2. Choice of mRNA substrate The choice of substrate (endogenous or exogenous) depends on the experimeutal question being asked and on various parameters such as the number of available cells, the amount per cell of the mRNA of interest, and the activity of 'nonspecific' cellular RNases (Table 2). NB. The term non-specific RNase denotes an activity capable of degrading all RNAs (rRNA, tRNA, or mRNA) at sim1Jar rates in vitro. The term is not meant to imply lack of function for such an RNase. only that it is not yet understood what its function might be. 108

Jeff Ross Table 2 . Use of endogenous and exogenous substrates for in vitro mANA turnover stud1es Endogenous substrate (mA NP, either free or polysome-associated)

Adltantages • ' authentic' substrate, made by cellular transcription :modification enzymes e known to be respons1ve to at least some trans-acting regulatory factors in vitro

Oisi1dvantages • d ifficult to detect 1f the mANA is scarce • prone to degradation during isolation • cumbersome when variants need to be analysed Exogenous substrate (protein-free cellular mANA or m vitro synthesized RNA)

Adv·anrages • easy to detect, especially if radiolabelled • ectsy to prepare variants

Disadvantages • translated w ith low efficiency in some extracts • m ight not respond to regulatory factors

2.1 Endogenous substrates (free mRNP or polysome-

associated mRNP) Polysomes contain the substrate messenger ribonucleoprotein (mRNP) to be analysed and often the messenger RNases as weU. There are several advantages to using endogenous mRNP substrates. First, the nature of the substrate is relatively clear, i.e . the mRNA was synthesized by the celJ and is competent for translation, since it is polysome-associated. Therefore, within the constraints imposed on any subcellular fraction prepared in the laboratory, an endogenous subst rate could be considered more or less authentic. Second, some endogenous substrates respond in vitro to factors involved in regulating mRNA stability (3-7). ln contrast, the turnover of some exogenous, deproteinized mRNA substrates is unaffected by such regulatory factors (see Section 2.2). There are three potential disadvantages of endogenous substrates. First, if th•e mRNA is scarce, large amounts of polysomes might be required to deteC't it. Second, polysomes containing undegraded mRNA can be difficult to prepare, especially from some primary animal tissues, unless special techniques are devised to red uce non-specific RNase activity (8). Third, it might be difficult or impossible to engineer cells to express interesting modified substrates, for example, mRNA that has a discrete 3 ' terminus and that lacks a polr(A) tract. 109

4 : mRNA turnover in cell-free extracts

2.2 Exogenous substrates The assa} is relatively easy with an exogenous substrate, especially if it is radiolabelled. The substrate is simply incubated '' ith a cell extract, purified. and analysed by gel electrophoresis. Exogenous substrates can be modified more easil} than endogenous mRNAs, "ithout concern for the effects o; the modifications on transcription, capping, or transport to the cytoplasm For e\ample, substrates of almost any size and sequence can be generated rapidly by cutting a eDNA clone at different sites and transcribing each template '' ith an appropriate bacteriophage RNA polymerase. HO\\e\'er, there are two important disadvantages of exogenous substrates. First, mRNA turnover and translation are often linked (I, 9, 10), but it ts difficult to translate exogenous mRNA efficiently in extracts from nucleatea mammalian cells. Second, some exogenous substrates, unlike their endogenou~ counterparts, fail to respond to regulatory factors in 1•itro. For example. polysome-associated (endogenous) c-myc mRNP is regulated in vitro by cytosolic destabilizers (3, 7) but deproteinized (exogenous) c-myc mRNA is not (G. Brewer and J. Ross, unpublished observations).

3. Preparation of undegraded mRNA and polysomes When using an endogenous substrate, it is important that the mRNA and polysomes prepared are undegraded in order to obtain meaningful data. Contamination of glassware or reagents with RNases, which seem to be ubiquitous, can ruin a preparation . The fo llowing precautions, coupled with a large dose of common sense, will help maintain RNA integrity: (a) Choose the proper cell or tissue. Some cells contain more non-specific RNases than others, so even the most careful investigator might have difficulty isolating intact polysomes from them . In general, tissue culture cell lines contain less RNase acrivity than animal tissues. If necessary, various RNase inhtbitors are available. Two of them, placental RNase inhibitor and diethylpyrocarbonate (DEPC), are discussed in this chapter. Vanadyl ribonucleoside complex at 4 mM and heparin at 0.2 mg/ ml have also been exploited to block RNase activity during preparation of extracts from anmtal tissues (8). (b) Use sterile plasticware whene,er possible. (c) Wear disposable plastic gloves at all times, since bod} surfaces and tluids comam active RNase(s). (d) Pre-rinse all glassware with DEPC at least one day before use (see Protocol 1). DEPC treatment is recommended, because autoclaving migdt not inactivate all RNase contaminants in the glassware. Moreover, DEPC is an excellent sterilizing agent. 110

leff Ross (e) Filter all solutions and store them at 4 oc or at - 20 °C. MagnesiUm iom, and carbon sources (e.g. sucrose) might permit growth of certain microorganisms, all of which contain RNases. The best way to handle these solutions is to sterilize them by filtration through a micropore ( ~0.451Jm) membrane (e.g. Millipore HA\\ P) in a tissue culture hood. Store them m a refrigerator, only open the stock bottles in a sterile hood, and dispense them using Sterile pipettes. Tape a note on each bottle stating that the solution is sterile and must be opened only in a hood. Some laboratories pretreat their glass-distilled water by adding DEPC to a final concentration of 0.02~o and then autocla,·ing. HPLC-grade ''ater or \\ater prepared by deionization and double distillation might be suitable for use without sterilization or DEPC treatment. (f) Carry out all procedures at 2-4 °C, from the time the cells are hanested until the extracts are placed in the - 70 oc freezer.

Protocol I describes the treatment of glassware '' ith DEPC and b> baking. Protocol 1 . Treatment of glassware to inactivate RNases.

1. Add a small volume of DEPC to each 1tem of glassware. 2 . Rotate the glassware so that all inner surfaces are exposed to the DEPC. 3 . Caution . DEPC can form a carcinogenic compound by reacting w1th primary amines. Therefore, after rinsing the glassware, pour the DEPC into a beaker

in a hood, add excess water, and then let the liquid evaporate to dryness. 4 . Rinse the glassware thoroughly with distilled water until all traces of the distinctive DEPC odour have d1sappeared (a min1mum of ten rinses should do). 5 . Bake the glassware in a 220 °C oven for at least 1 h, preferably overnight.

4. Preparation of cell extracts for in vitro mRNA

decay reactions There is no single, universally accepted standard method for analysing mRNA turnover in vitro, nor is one likel} to evolve. Different e>."tracts and reaction conditions are required to account for variables which can influence mRNA turnover within the cell. These variables, in turn, depend on the hormonal and nutritional status of the ceil, its position m the cell cycle, and its stage of differentiation. For example, the half-lives of some hepatic cell mRNAs can fluctuate by 30-fold or more when certain hormones are added to or removed from the culture medium. The following questions are relevant to the stability of most mRNAs and must be considered in deciding how best to carry out cell-free mRNA decay reactions. 111

4:

e Is

mRNA turno,·er in cell-free extracts

the mRNA translated in cells and, if so, at what rate?

• Does the cell contain trans-acting regulatory factors which inOuence the mRNA half-life? If so, are these factors freely diffusible throughout the cytosol or are they associated with particular cytoplasmic components such as ribosomes, endoplasmic reticulum, the mRNA itself, etc.?

e Is the mRNA itself localized as a result of its association with microfilaments. endoplasmic reticulum, etc.? Three methods are described for preparing extracts. Each has advamagt and disadvantages, depending on the mRNA to be analysed and the variable' cited above. The investigator must choose the appropriate extract or combination of extracts, substrates, and in vitro conditions so as to reOect as closeh as possible the circumstances under which the mRNA is degraded withtn the cell. The first method exploits ultracentrifugation to prepare polysomes and post-polysomal supernatant (Sl30) from cells homogenized in a detergent-free, low-salt buffer (see Protocol 2). A major advantage of this method is us nexibility. Since polysomes contain mRNA and messenger R 'ase(s), t '1e turnO\er of polysome-associated mRt As can be analysed simpl}' by incubati 1g polysomes under appropriate conditions (see Protocol 6). The S 130 seems to ha\e little messenger RNase activity but does contain soluble, trans-acting factors which regulate the half-lives of certain mRNAs. These factors can be assayed in reactions containing mixtures of SJ30 and polysomes (see Protocol 6 . Exogenously added, radiolabelled mRNA can also be assayed in reaction~ containing polysomes or polysomes plus Sl30 (see Protocol 7). Ribosome-bound messenger RNases can be separated from polysomes by exposing the polysomes to high salt, pelleting them in an ultracentrifuge, and harvesting the supernatant (see Protocol 3). The high-salt extract can then be used to analyse the decay of radiolabelled mR A (see Protocol B). One disadvantage of using detergentfree polysomes is a very lo\\ translation rate, both for endogenous and exogenou' mRNAs. The second method for preparing extracts uses lysolecithin to permeabilize adherent ussue cullure cells, which are then scraped from the culture di~h. broken, and brieO) centrifuged to remove nuclei (see Protocol4). These extra ts are easy to prepare, and they support translation of endogenous and exogeno ts mRNAs more efficiently than detergent-free extracts (see ProTocols 9 and 10 ). One disadvantage is a lack of information on whether lysolecithin extracts retain their translational and mRNA turnover activities \\hen they are fractionated or frozen. The third method u~es a lysate prepared from reticuloc)1e , \\hich arc immature. enucleated red blood cells contairung abundant polysomes (see Protocol 11 ). This system is the most active of the three for translation, and reticulocyte extracts can be purchased at modest cost. The lysolecithin and reticulocyte extracts can be made mRNA-dependent by exposing them brieOy 112

]eft Ross to micrococcal nuclease, which eliminates the mRNA but not the ribosomes or other components necessary for translation (see Protoco/5). After inactivating the micrococcal nuclease, the extract can be incubated with exogenous mRNA under conditions permitting active translation and the half-life of mRNA which had become polysome-associated can be determined (see Protocols 10 and 11). Micrococcal nuclease treatment of detergent-free extracts is fruitless, because translation initiation occurs so inefficiently. It is important to stress once again that these methods should be used as guides and may need to be modified to suit specific experimental parameters. Flexibility is essential and extracts can, in theor}, be mixed in various combinations. For example, if an investigator wishes to analyse the turnover of a mRNA which is being actively translated, radiolabelled mRNA can be incubated in nuclease-treated reticulocyte extract. However, suppose that the investigator suspects that the intracellular half-life of this mRNA is influenced by a cytosolic trans-acting regulatory factor, which is likely to be presem in S 130. Since reticulocytes are end-stage cells whose S 130 contains primarily haemoglobin, they are unlikely to have retained all of the S 130 regulatory factors which are present in undifferentiated, nucleated cells. Therefore, the best approach might be to mix the mRNA to be tested with reticulocyte extract plus S 130 isolated by detergent-free lysis of nucleated cells. The multicomponent reaction mix might reflect the intracellular conditions to which the mRNA is normally e'posed (efficient translation plus a soluble regulatory factor) more accurately than reactions containing only detergent-free polysomes (minimal translation, no soluble factors) or detergent-free polysomes plus S 130 (soluble factors present, but minimal translation). Protocol 2 and the following protocols pertain to tissue culture cells, since limited information is available on the use of extracts from animal tissues for cell-free mRNA decay. In fact. it is unclear whether these protocols can be used with primary animal tissues. Investigators wishing to use animal tissues should take special care to reduce non-specific RNase activity. The following RNase inhibitors might be useful: • Placental or liver RNase inhibitor (II), available from Amersham, Boehringer Mannheim, Calbiochem, ICN Biomedicals, Promega, Stratagene. Useful amounts must be determined empirically, but an appropriate starting point is 100 units per mi. • Ribonucleoside-vanadyl complexes (12), available from Bethesda Research Laboratories, Ne\\ England Biolabs, Sigma. Starting point: 2.5 roM. Moreover, buffers can be designed to contain combinations of inhibitors which might be more effective than a single inhibitor. The composition of one buffer used to isolate intact polysomes from primary cultures of amphibian liver is given below (8): 113

4: mRNA turnover in cell-free extracts • 0.2 M sucrose e 70m~1 KCI e IOmM MgCI2 e SmM NaCl • 4 mM vanadyl-ribonucleoside complex e l mM EGTA • 0.2 mg/ml heparin el mM DTT • 100 units/ml placental RNase inhibitor e SO mM Hepes-NaOH, pH 7.8

Protocol 2 . Preparation of polysomes and post-polysomal supernatant (S 1 30) from exponentially-growing tissue culture cells lysed without detergent8

Equipment and reagents NB All buffers and tubes should be at 2-4 °C prior to the experiment pestle with a clearance of approximately 0.1 mm; use a vessel with a capacity 2to 3-fold greater than the volume of Solution A used for cell lysis (see step 6 1; a Dounce-type glass homogenizer with a tight-fitting pestle (clearance 0.06 mml may also be required (see step 17)

• Exponentially-growing tissue culture cells • Solution A (1 mM potassoum acetate, 2 mM magnesium acetate, 2 mM DTT, 10 mM Tris-acetate, pH 7.6 at room temperature) • Solution 8 (solution A containong 30% (w/v) RNase-free sucrose) • Tissue culture medium without serum or antibiotics; medium Ham's F 12 (Flow, Gibco, HyCione) has been used in the author's laboratory, but any osotonic growth medoum should do • Potter-Elvehjem homogenozer with Teflon

• Low-speed centrifuge, ultracentrifuge, and appropnate tubes; polyallomer ultra centrifuge tubes are preferable; they need not be baked but should be thoroughly rinsed with RNase-free water and then drained dry immediately pnor to use

Method 1. Count the cells. 2. Centrifuge the culture for 4 min at 34 g (average I. corresponding to 500 r.p.m 1n a low-speed centrifuge with a swinging bucket radius of 8 em. Note: This and all subsequent procedures should be performed at 1-2 ° C. All buffers should be kept ice cold throughout the manipulations. 3. Asporate and discard the supernatant. 4. Gently resuspend the cells in ice-cold tissue culture meoium without serum or antibiotics. Centrifuge as in step 2. 5. Repeat steps 3 and 4.

Continued 114

Jeff Ross Protocol 2 . Continued 6 . Resuspend the cHIIs in solution A. The volume will depend primanly on the cell number and the cell type, since some cells do not lyse easily by mechanical homogenization, especially if they are too concentrated (see step 8 below) . The maximum concentration of cells for high· efficiency homo~Jenization (95% or greater lysis) must be determined in a pilot experiment by counting the percentage of broken cells using a haemocytomet er and microscope. In most cases, 10 8 cells/ml is a reasonable starting point, but the optimum number might be up to 10-fold lower. 7. Transfer the cells to the prechilled Potter-Eivehjem homogenizer.

8 . Place the homogenizer vessel in an ice-water bath and break the cells by moving the pe!stle rapidly up and down and twisting it left and right at the same timc3. The number of up and down strokes required for efficient cell lysis will vary, depending upon the cell type and their concentration. As a starting point, 20 strokes should do (see step 6 above). Avoid generating excess bubbles. 9 . Centrifuge the homogenate for 10 min at 8300 g (average ), which corresponds to 9000 r.p.m. in a swinging bucket rotor, radius 9.1 em. 10. Remove and save the supernatant (S 10), taking care to avoid the pelleted material (nuclei and membranes). It is best to leave behind a small amount of supernatant so that none of the pelleted material contaminates the S 10. 11 . Gently layer the S 1 0 over a cushion of solution B in an ultracentrifuge tube. The volume of solution B should be approximately 25% of the tube capacity. If the tube is insufficiently filled with S 10, add solution A.

12. Centrifuge at 130 000 g (average). 2 °C, 2. 5 h, which corresponds to 36 000 r.p.m. in a Beckman SW60 rotor. If you intend to save the S 130, allow the rotor to decelerate without the brake. 13. Carefully remove! the supernatant (S 130) above the sucrose cushion, taking care not 1ro harvest the whitish material located on top of the cushion. Leave a small amount of S 130 above the cushion, if necessary. Do not be concerned if some of the lipid at the top of the tube is harvested along with the S 130. Set aside the S 130 in an ice bucket. 14. Aspirate the remaining liquid, including the sucrose, and discard it. A bluish-white pellet of polysomes should be visible in the centre of the tube bottom. 15. Lay the centrifuge tube on its side and cut it across with a razor blade approximately two-thirds from the top. Discard the top piece.

16. Gently add approximately 1 ml of solution A down the sides of the bottom part of the centrifuge tube. Gently move the tube back and forth to wash any contaminating matenal, including sucrose, from the upper surface of the polysome pellet. Aspirate or pour off the liquid. Repeat this washing step once. It is not necessary to perform these washing steps quickly, since the polysomes will not dissolve in the buffer, but it is necessary to add the buffer slowly and gently, so as not to dislodge the polysome pellet from the bottom of the tube.

Continued 115

4: mRNA turnover in cell-free extracts Protocol 2. Continued 1 7 . Resuspend the polysomes in a small volume of solution A . The volume will depend on the quantity of polysomes recovered and the subsequent experiments. As a rule. use a volume such that the polysomes from 10 6 cells will be resuspended per microlitre. Polysomes can be difficult to resuspend. They tend to be 'sticky', forming clumps that are difficult to disperse. First, try resuspending them by pipetting the liquid up and down, using an automatic pipettor with a 200 1-11 or 1 ml disposable tip. If visible clumps remain, transfer the material to a tight-fitting Douncetype glass homogenizer with a clearance of 0.06 mm. Several up and down strokes with this homogenizer should break up the clumps. 1 8 . Measure the optical density of the polysomes and S 130 at 260 nm and 280 nm and store both preparations in multiple aliquots at -70 °C. As a guide, polysomes isolated from a logarithmically growing human erythroleukaemia cell line (K 562) resuspended at 10 6 cells' polysomes per microlitre give an absorbance reading of 220-260 through a 1 em light path at 260 nm.

19 . To assess polysome integrity, RNA from a small amount of the preparation can be extracted and analysed by agarose gel electrophoresis (13. 14). The rRNA can be visualized by ethidium bromide staining ( 13, 14). The rRNA is intact if the 28S and 18S rRNA bands are discrete and if the band intensity appears to be approximately 2:1 (28S: 18Sl. • from refs 13, 14.

Protocol 3 . Preparation of ribosom al salt wash (RSW) from det ergent-free polysomes8

Equipment and reagents • Polysomes isolated as in Protocol 2, steps 1-16

• 4 .0 M potassium acetate

• Solutions A and B. and ultracentrifuge equipment as in Protocol 2

Method 1. Resuspend the polysomes in solution A. The polysomes need not be fully dispersed, as described in step 17 of Protocol 2, but they should be more concentrated. A reasonable starting point is 2-5 x 109 cells' polysomes per millilitre. but higher concentrations can be used, if desired.

2. Transfer the polysomes to a small test tube or beaker containing a stirring bar, in the coldroom. Prior to use, the stirring bar should be soaked for 3-5 min in DEPC and rinsed thoroughly with water. 3. Add sufficient 4.0 M potassium acetate dropwise and with gentle sttrring to bring the final concentration to 0.5 M.

Continued 116

jeff Ross Protocol 3 . Continued 4 . Stir gently for an additional 1 5 min in the cold. Avoid bubbles.

5 . Layer the material over an appropriate volume of solution B in an ultracentrifuge tube and centrifuge for 2. 5 h at 130 000 g (average). 2 ° C. 6 . Carefully harvest the RSW located above the sucrose cushion. Retain the salt-washed polysome pellet (see step 8). 7 . M ix it gently but thoroughly, determine the protein concentration b ( 15), and store the RSW in small aliquots at - 70 °C. 8 . If desired, the salt-washed polysomes in the pellet at the bottom of the tube (step 6) can also be harvested, as per Protocol 2 , steps 16-19. ' From ref. 14. b Kits available from Bio-Rad and Pierce contain reagents for the Bradford protein determination assay.

Protocol 4 . Preparation of crude lysate by homogenization with lysolecithin8

Reagents • Non-confluent. adherent tissue culture cells in 100 mm diam. tissue culture dishes; suspension cells can perhaps be used by substituting low-speed centrifugation and gentle resuspension for aspiration (see step 4 below) • Solution C (0. 15M sucrose, 33 mM NH 4 Cl , 7 mM KCI, 4.5 mM magnesium acetate, 30 mM Hepes- KOH , pH 7.4) • Solution D ( 100-300 IJg/mllysolecJthin (L-a-lysophosphatidylcholine, palmitoyl) in solution C )0

• solution E (0.2 M NH 4 CI, 20 mM magnesium acetate. 7 mM KCI, 1 mM OTT, 1 mM A TP (dipotassium salt), 1 mM GTP (sodium salt). 40 11M each of the common 20 amino acids, 0.1 mM 5 -adenosylmethionine, 1 mM spermidine, 10 mM creatine phosphate (dipotassium salt ), 40 units per ml of creatine kinase, 100 units of placental RNase inhibitor, 0 .1 M Hepes-KOH. pH 7.4)•

Method 1. Aspirate the medium from each dish of cells.

2 . Place the dish on ice and quickly wash the cells twice with 2- 3 ml icecold solution C, removing the liquid by aspiration. 3 . Add 1- 2 ml of solution D to the dish and leave for 60 sec on ice. Be sure that the entire surface of the dish is covered w ith liquid. 4 . Aspirate the solution, and gently invert the dish for 60 sec to drain the remaining liquid to one corner.

5 . Aspirate off the remaining solution D. 6 . Put the dish back on ice and add 200111 of ice-cold solution E. 7 . Scrape the cells from the dish with a standard rubber-tipped cell scraper and transfer the cell suspension to a microcentrifuge tube.

Continued 117

4 mRNA turnover in cell-free extracts Protocol

4 . Contmued

8 . Disrupt the cells by passtng them 1 0 times through a 2 5-gauge needle attached to an appropnately-sized hypodermtc syringe. Monttor cell lysis by mtcroscopy. at least lor the first few ttmes the extraction is performed. If asptratton mrough a needle appears to be too harsh (i.e if the nuclei are disrupted cells can also be lysed by aspirattng them repeatedly with a Pasteur ptpette.

9 . Centnfuge the cell lysate tn a mtcrocentnfuge in the coldroom for 1 min to pellet the nuclei.

10. Remove the supernatant. taktng care not to contamtnate tt Wtth pelleted material.

1 1 . Store the supernatant on ice in preparation for m VItro mANA decay reacttons. It may be possible to store the lysate in aliquots at - 70 °C but the effects of freezing and thawing on the translational or mANA turnover activity of these extracts have not been reported. Therefore, preliminary studtes mtght be required to determtne whether freeztng ts feasible. • From ref. 5. • The concentration of lysolecithm to be used depends on the type of cells and must be determtned emp,ncally, since excess lysolectthtn will reduce translattonal activity. while msufftcient l¥s01ectthin wtll not permeabilize the cells. Brown et a/. ( 161 used 100 ~g :ml for PK ce !s, a pig kidney hne, and 250 IJg/ml for BHK cells, a baby hamster kidney line. Krikonan and Read (51 used 300 ~g/ml for Hela cells. Try different concentrations and compare the capacity of each extract to translate endogenous mANA, using the method descnbed 1n Section 5.2 < The opttmum concentrations of magnesium, potassium. and ammonium tons for translation or mANA turnover seem to vary considerably depending on the source of the cell extract and so should be determined for each cell line (dtscussed in ref. 161. The concentrations described here have been opttmized for Hela cell extracts (51. Opttmum concentrat the source of endogenous mRNA. 119

4: mRNA turnover in cell -free extracts r------------------------------------------------------------~

Protocol 6. Turnover of endogenous polysomal m ANA in detergentfree extract s 8 Reagents e Solutton F. prepare this as follows and store ot on small aliquots at - 70 ° C

Component

Final concentration in 2~i 111 reaction mixtures

Ill

water

200

1 0 M creaune phosphate

200

10mM

40

2mM

1MDTI 0 . 2 M A TP (Tris salt)

100

1 mM

0 .2 M GTP (Tris salt)

20

0. 2mM

2.0 M potassium acetate

01 M

1000

2. 5 mM spermine

800

0.1 mM

1 0 M Tros- acetate. pH 7.5

200

10 mM

• Solution A and polysomes at a concentration of 106 cells' polysomes ~o~l prepared as described m Prorocol 2

solution F must be added before the RNase inhibitor; the volumes goven are for each 25 ~o~l reactoon requored in step I

• Creatine krnase (4 mg/mlon 50% glycerol)

solution F

• 0 . 1 M magnesium acetate

4 mg/ml creatine kinase

0.2 5~o~ l

• Placental RNase inhibitor (40 unots l~o~ll

RNase inhibitor

0 .25111

• Solution G prepare this solution on oce JUSt prior to performing the reactoon; the author does not know if the order of additoon of components is omportant, but

solution A

3.2~o~ l

161JI

0 . 1 M magnesium acetate

0 . 2111

water

1. 1 Ill

Method 1 . Add the following to the required number of microcentnfuge tubes on ice • solution G

21 ~o~l

e polysomes 2 . Mix gently without vonexong and incubate the reactions at an appropriate temperature, usually 37 °C , for the required t ome. No smgle reactoon temperature can be considered optimal f or all mRNA decay reactions. Therefore, while standard reactions are usually incubated at 37 ° C, Situations may arise in whoch it may be advantageous to incubat•~ at lower temperatures. For example, cell -free mRNA turnover reactions are panicularly useful for odentofying mRNA decay products. The nature of these products provides a pict ure of t he decay pathway: 1ts direction (5'- 3' or 3 ' - 5'1 and t he sons of enzymes involved (exo- or endo ribonucleases). However, decay products of some mRNAs might be too unstable to be detected even in vitro at 37 ° C . If so, try incubating the reactions at a temperature between 20 ° C and 32 ° C , rather than 37 °C . React ion rates are slower at lower t emperatures, permotting easier detection of decay products (17).

Continued

120

Jeff Ross Protocol 6. Continut?d 3 . Remove the tubes .and either store them at - 70 °C or extract the RNA immediately for analysiS. Any RNA extraction method can be used, as long as care is taken to avoid non-specific RNase contamination. RNA extraction methods are g1ven 1n Volume I, Chapter 1 From refs 13. 14.

As mentioned in Sections 4 and 5.1, various combinations of subcellular fractions can be assayed in this system, which is one of its important features. The components of the reaction mixture given in Protocol 6 (step I) can be modified accordingly. For example: (a) Some trans-acting factors that regulate mRNA stability are located in the S 130 fraction and can be ido~ntified by mixing Sl30 and polysomes. Simply substitute Sl30 for an equivalent volume of solution A when making up solution G. To assay the effect of I0 ~I of S 130 on the decay of polysomal mRNP, prepare solution G with 6 ~I per reaction of solution A, instead of 16 ~1. and set up reactions containing I 1 ~I of solution G, 10 ~I of Sl30, and 4 ~I of polysomes. (b) To analyse mRNP decay in SIO, simply substitute SIO for polysomes and solution A. Care should be taken to maintain the magnesium ion concentration at 2 mM.

5.1.2 Using exogenous RNA substrates A system for assaying the turnover of exogenous RNA in detergent-free extracts is described in Protocol 7. The method is similar to that described in Protocol 6, but the substrate is protein-free (phenol-extracted) RNA, not endogenous polysomal mRNP. The two major sources of substrate are cellular mRNA or RNA synthesized in vitro. Cellular RNA should be prepared from a cytoplasmic extract and can be pre-purified by affinity chromatography to select poly(A) + mRNA. If synthetic mRNA is used as a substrate, some important \ariables must be considered. Should the mRNA be synthesized in the presence of a cap analogue (m-GpppG)? Should it be polyadenylated? Should it be radiolabelled, and, if so, to what specific activity? As a general rule, it seems reasonabl.e initially to use a substrate as close as possible to authentic, i.e. both capped and polyadenylated. Protocol 7. Turnover of exogenous mANA substrates in detergentfree extracts 8

Reagents • SolutiOn A and polysomes 1105 cells' polysomes/IJI as described in Pro-

e creatme kinase, RNase 1nhib1tor, solutoon F, magnesium acetate as described in

toco/2

Protocol 6

Continued 121

4: mRNA turnover in cell-free extracts Protocol

7. Continued

• E•ther a ' P·•abelled RNA substrate 1- 107 c. p .m . IJQ, 5000- 20 000 c.p .m. l ~o~ l m w aterl or unlabe lled RNA 10 .1 -2.0 ~o~g . ~o~l rn water; se•:1 Volume I, Chapter 1 !Protocols 3 and 4 1 and Chapter 4, th1s volume (Pro tocol 4 I

• Solution H: prepare thrs on ice; the

formula below •s fo r each 25 ~o~l reactJOn i n step 1: solut1on F 3 . 2 IJI 4 mg ml creat1ne kmase 0 .251JI RNase mhibrtor 0 .251JI solution A 161JI 0 . 1 M magnes1um acetate 0 . 21JI RNA substrate 1 liJI reQu ~red

Method 1. Add the following to thH required number of mrcrocentrifuge tubes on ice· • solution H e polysomes

21

~I

2. Incubate the reactions at 37 °C for the des1red t ime. 3. Erther store the reaction mrxtures at - 70 ° C or extract the RNA immediately (see Protocol 6 , step 3 ). • From refs 1 3, 14.

The reaction mixtures described in Protocol 7 can be modified to incluue SIO, Sl30, or combinations thereof. When doing so, the composition of solution H must be changed, decreasing the volume of solution A o accommodate that of the added subcellular fraction. If it is necessary to add more than 2 ~g of RNA substrate per 25 ~I reaction, preliminan assays should be performed at the higher substrate levels, since excess R '.. \irus, the other was 'mock infected'. meaning that it was treated in the same way but no virus was added. • At 60, 180, and 360 min following virus inoculation (mixing virus and cells). cells were harvested and post-polysomal supernatant (S 130) "as prepared. as per Protocol 2.

• In vitro mRNA decay reactions were performed by mixing polysomes from uninfected cells wnh Sl30 from either mock-infected or "irus-mfected cells, as per Protocol 6. • Each reaction was incubated for 0 to 150 min; then total RNA was isolated and annealed with a 31P-labelled DNA probe for human -y-globin mRNA. The amount of undegraded ,-globin mRNA in each reaction was determined by mcubation with nuclease Sl and electrophoresis of nuclease-resistant DNA in a gel. 129

4: mRNA turnover in cell-free extracts

Mock in V//ro

0 15 30 90 150

Incubation T1me (min)

Y-Giobin-.

Wlld-type Post-Inoculation Time(min)

180 o

15 30 90 150 0

15 30 90 150 0

360 15 30 90 150

In vitro

< lncubat1o1 nme(m•

~

Y-Giobin -.

Figure 3. Identification of an mRNA destabillzmg factor 1n the S 130 of cells mfected w1th Herpes s1mplex v1rus. Tissue culture cells were either mock -infected or infected with Herpes Simplex virus for up to 6 h, as described in the text. S 130 was prepared from each culture flask and was added to polysomes from uninfected cells. The react1ons were Incubated as per Protocol 6. The stability of polysome-associated -y-glob1n mANA was determined by nuclease S 1 mappmg. Top panel, polysomes plus S 130 from mock-infected cells, 1 e. cells not exposed to virus. Bottom panel, polysomes plus S 130 from cells infected for the indicated times [wild-type, post-moculation time (min) I with Herpes s1mplex v~rus . The time course 1n mm of each incubation is shown {in VItro •ncubation time lmml I above each lane. Repnnted with permiSSIOn from ref. 6

Figure 3 shows that polysomal, endogenous )-globin mRNA is ver} stable in reactions supplemented with SJ 30 from mock-infected cells (no virus) but that S 130 from cells infected with the virus for 60, 180, or 360 min contains a potent mRNA destabilizer. Th.is destabilizer is now knO\\n to be a protein encoded by the' irus and an integral part of the virion itself. By an unknown mechanism. this protein accelerates the degradation of almost all cellular mRNAs, even tho,e like )-globin which are normally very stable in uninfected cells.

6.2 Data interpretation As discussed in Section I and Protoco/6, mRNA decay rates are slower in ,.;rro than in intact cells. Relatively slow reaction rates are also observed in cell-tree 1 30

Jeff Ross transcription and translation systems. Therefore, it seems reasonable to exploit cell-free mRNA decay systems to measure relative, as opposed to absolute, decay rates. To assess whether the system is functioning properly, the decay rates of mRNAs which are known to have different intracellular half-lives should be determined. mRNAs that are unstable in ceiJs should also be unstable in vitro, relathe to stable mRNAs. Some caution must be exercised in plotting and interpreting in vitro mRNA decay data. In cells, mRNA is degraded in a logarithmic fashion, according to the formula:

where C and C 0 are mRNA levels at time t and time 0, respectively, and kd is the decay constant (reviewed in ref. 25). The problem with applying similar parameters to in vitro data is that decay rates may decrease as the reaction time increases. For example, the author has found that the c-myc mRNA decay rate decreases at least two-fold after several hours of incubation, presumably because critical reaction components become rate-limiting (J. Ross and G. Brewer, unpublished observations). Therefore, it seems reasonable to plot the in vitro data as either a linear or logarithmic function , provided that each mRNA is analysed by the same criteria (see Figure I as an example).

6.3 Troubleshooting Several potential obstacles and trouble spots which might arise during the course of cell-free mRNA decay experiments are listed below, as well as some suggestions for solving them.

6.3.1 Excessive RNA degradation during preparation of polysomes and extracts Precautions for reducing endogenous, non-specific RNase activity while preparing polysomes and during in vitro incubations are discussed in Protocol 2. If these are inadequate, it might be helpful to avoid using primary animal ceiJs and to use instead a tissue culture cell line synthesizing the mRNA{s) of interest. Some cells lines contain less 'non-specific' nuclease activity than others. Try screening several ceiJ lines to find one which allows intact polysomes to be isolated.

6.3.2 All mRNAs in the mRNA decay reactions are degraded at similar rates Again, non-specific RNase activity might be overwhelming the system. Se"eral modifications should be tried: • Add more RNase inhibitor or try various combinations of inhibitors. 131

4: mRNA turnover in cell-free extracts • Modify the reaction conditions. For example, non-specific RNase acti\ il} might be depressed by changing the mono' alent or divalent cation concentrations, the pH, or the reaction temperature. • Add carrier single-stranded RNA, for example, poly(C) at 1-21Jg 25 ~ reaction (see Protocols 6-8). Avoid higher amounts of exogenous RNA, whkn can slow mRNA decay processes.

6.3.3 No mRNA degradation is observed The simplest explanation for this result would be inactivation of messenger RNases (and other RNases) during extract preparation. If no RNA is degraded during in vitro reactions, the composition and pH of all extraction buffers should be monitored carefully. An unlikely explanation is that all RNase activity tn the extract is blocked by the placental RNase inhibitor. Therefore, reactions should be carried out without inhibitor.

6.3.4 Regulated decay is not observed For example, a particular mRNA might be known to be degraded five-fold It ~s rapidly in cells treated with a translational inhibitor, as compared with untreated cells. However, the mRNA is found to be degraded in vitro at the same rate with polysomes isolated from treated and untreated cells. There could be many explanations for this result, but the following can be investigated: • Add S 130 (Protocol 2) to the reactions. Perhaps the regulatory factor is n the S130 and is not polysome-associated. • Alter the reaction conditions, as per Section 6.3 .2. • Prepare a lysolecithin extract (Protocols 4 and 9) rather than a detergentfree extract from both sets of cells, to encourage translation of the mRN A., since the regulation of some mRNAs might be linked to their translation. In this case, the mRNA might be unstable under conditions of efficient translation elongation, but stabilized when elongation is retarded by an inhibitor. The in vitro system would then reflect the intracellular situation only by comparing the stability of elongating versus non-elongating mR}'I. '\, Some translation does occur in the lysolecithin extract but little is obsen t.'ol, 30% glycerol. 6% SOS, 0. 1% bromo phenol blue) • SDS- PAGE gel 110% acrylam•de prepared accord1ng to Volume I. Chapter 5. Protocol 7 or ref 371

Method Preparation of the pre-rRNA substrate substituted with 4 -S-UMP

1. Synthesize the pre-rRNA substrate substituted w1th 4 -S UMP by setting up the following reaction (in the order given) in a microcentrifuge tube on ice:

• H2 0

volume to achieve 48 5 Ill final volume

• 5 x transcription buffer e o 2M OTT

10~JI 2~JI

Contmued

162

Cathleen Enright and Barbara Sol/ner-\Vebb Protocol 13. Continued • RNastn

21JI

e 5 mM A TP GTP solutiOn

51JI 4.51JI

e 10mM CTP e 2 mM 4 -S-UTP e [a-32P] CTP (120 llCil

51JI 121JI

• template DNA (0. 7 IJgl

31JI

2. Add 1. 51JI of T7 RNA polymerase and mcubate the reaction at 37 ° C for 90 mm, keeping 1t as light-free as poss1ble. 3 . Isolate the 32P-Iabelled, substituted rRNA product by electrophoresis on a sequencing gel, as described in Protocol 3, steps 2-7 4. Calculate the yield of rRNA product as described m Protocol 3, steps 8-12, using UTP as the marker nucleotide. Preparation of processmg complexes using 4-S-UMP substituted pre-rRNA substrate

NB Protect the process1ng react1ons from light m steps 5-7 to mm1m1ze spurious cross-linking. 5 . Set up a 50 IJI processing reaction, as described in Protocol 5, (step 1) by mixing : • 4 -S-UMP substituted rRNA (step 3) • S· 100 processing extract e 5mM ATP

volume contaming 10 fmol 31JI

• extract buffer • 5 x S· 100 processing buffer (With NaCI in place of KCI)

10111

• H2 0

to 50 IJI fmal volume

6 . Incubate the reaction for 50 min at 30 °C and then place 1t on ice 7 . Add 2 Ill of 20 IJg/ml heparin and incubate the mixture for an additional 10mm at 30 ° C. Cross-/inkmg of processmg protems to rRNA and SDS-PAGE analysis 8 . Transfer the sample to one of the wells 1n a 96-well m1crot1tre plate and place the plate 1n a Pyrex dish contaming just enough cold water to allow the microtitre dish to float. 9 . Place the Pyrex d1sh on top of the UV transilluminator and irradiate the sample for 1 5 m1n using the analytical power settmg 10. In a m1crocentrifuge tube, m1x 40 IJg of RNase A and 200 units of RNase T1 tn H 2 0 to a final volume of 20 IJI. 11 . Transfer 20 Ill of the irradiated processing sample (step 9) to the microcentnfuge tube containing the RNa se A and RNase T1 (step 10) and incubate the mixture for 1 5 min at 30 ° C

Continued 163

5: Ribosomal RNA processing in vertebrates

Protocol 13. Contmued 12. Add 20 111 of 3 x SDS-gelloading buffer to the digested sample and heat the m1xture for 3 mm at 95 ° C .

13. Load the sample onto the SDS-PAGE gel and separate the protems as descnbed 1n Volume I. Chapter 5, Protocol 7, or ref . 37 14. ldent1fy the proteins cross-linked to the nuclease-resistant 32P-Iabelled RNA residues by autoradiography. • Ba:sed on ref. 34.

Representative data from a UV cross-linking experiment are shown in Figure 4C. Polypeptides that become labelled when using a radiolabelled processingcompetent rRNA substrate, but not when using a radiolabelled processingincompetent rRNA substrate, are deemed to be specifically associated. Pre-incubation of the processing extract with unlabelled processing-competent or processing-incompetent rRNA substrates before the addition of the labelled 4-S-UMP substituted substrate affords confirmation of specificity. The subsequent labelling of specifically associated polypeptides will be aboli 11ed by the former but not by the latter competitor RNA as shown in Figure 4C.

5 . Assessment of the requirement for cellular RNA in

rRNA processing Section 4 described methods whereby processing polypeptides associated with pre-rRNA could be identified. Processing of pre-rRNA may involve the participation of several cellular RNAs, e.g. U3 snRNA. This section describes micrococcal nuclease treatment and oligonucleotide-directed RNase H digestion of RNAs in a processing extract which can be used to provide evidence for the requirement of cellular RNA in the processing reaction.

5.1 lMicrococcal nuclease digestion If treatment of a processing extract with micrococcal nuclease (but not a 'mock' treatment) destroys the processing acthrity, and this is not restored b) the addition of non-specific RNA, it can be presumed that the processing reaction has a requirement for one or more specific nucleic acids. Lack of sensu '>'ity to DNase I can further suggest that the nucleic acid is RNA. Note that an ab~e:1ce of an effect of micrococcal nuclease is not proof that an RNA is not reqw ed; a required RNA may not be accessible to the nuclease. Protocol 14 describes the treatment of the mouse S-100 processing extract with micrococcal nuclease. The advantage of micrococcal nuclease for this type of analysis is that it is a Ca2 + -dependent nuclease. This allows it to be inactivated once digestion of a putative nucleic acid is complete, using an excess of the Ca 2 + -chelator EGTA, so that the processing activity of the treated extract can then be tested. 164

Cathleen Enright and Barbara Sollner-\Vebb The length of time required for micrococcal nuclease digestion of the extract should be determined in a pilot experiment, but the time in Protocol 14 has been used successfully in the author's laboratory.

Protocol 14. Treatment of the mouse S-1 00 processing extract with micrococcal nuclease8

Equipment and reagents • S-1 00 process1ng extract (see Protoco/6) e 10mM CaCI2 • Micrococcal nuclease (BRL, 1 mg ml) e 60mM EGTA , pHS.O • Equipment and reagents for studymg

processing of pre·rRNA as described m Protocol 7

• Modified 5 x S 100 processmg butler (0.42 M KCI, 40 mM Hepes-KOH, pH 7.9, 16 mM MgCI2 , 33% glycerol 2.6 mM DTI. 0.46 mM EDTAI

Method 1. Mix 6 )JI of the S- 100 processing extract with 1 )JI of 1 0 mM CaCI 2 and incubate the mixture for 30 sec at 30 °C. Cool the reaction mixture on ice. 2 . Add 2 )JI of 1 mg/ml micrococcal nuclease and incubate the extract for 90 sec at 30 °C . Cool the tube on ice. 3 . Add 1 )JI of 60 mM EGTA and incubate for 30 sec at 30 °C. Again cool the extract on ice.

4 . Test the processing activity of the treated extract as described in Protocol 7, except replace the 5 1.11 of S- 100 processing extract with the 10 )JI of the nuclease-treated extract (step 3 above) and substitute modified 5 x S-1 00 processing buffer. • From ref. 34.

Data from an experiment in which the mouse S-100 extract was treated with micrococcal nuclease and then tested for its ability to process pre-rRNA are shown in Figure 6A.

5.2 Oligonucleotide-directed RNase H digestion of cellular RNAs involved in pre-rRNA processing Oligonucleotide-directed RNase H treatment can be employed to determine whether a particular RNA is required for a processing event. RNase H digests RNA only in an RNA:DNA hybrid. Therefore, hybridization of an oligodeoxyribonucleotide complementary to a specific RNA thought to be involved in processing could render that RNA susceptible to RNase H digestion. If this RNA is digested by RNase H (checked by Northern blot analysis) and is an essential component of the processing extract, the extract will then be unable 165

5: Ribosomal RNA processing in vertebrates

A

8

--

pl"oduct

' I

..

""c "

-

C. C.,E C.

-

...

...

•I

'f•12

UJ o

':!

....

0



2

~"----~

.... "'

OS

3

0

5

Figure 6 . Sens1t1v1ty of the mouse 5' ETS processmg reacuon to micrococcal nuclease or RNase H treatment of the mouse S- 100 processmg extract !AI Micrococcal nuclease sens1t1V1ty Samples of the mouse S- 100 processmg extract were treated as Indicated 1n tlle table below the autoradiograph. At the start It= 0), samples received CaCI2 (Ca; lanes 3 and 5) or CaCI2 and excess EGTA ICa.E; lane 4), or no add1t1on (lane 21 After 4 m•n It= 4Lmicrococcal nuclease IMN) was added (lanes 4 and 5). After another 4 m•n It =8), excess EGTA was added to all samples lackmg this reagent. Finally after a further 4 m1n It= 121 np. labelled pre-rRNA substrate was added to all the samples to test their processing ability The pre-rRNA (precursor) and processed product (product) were separated electrophoret•calty. lane 1 shows the untreated pre-rRNA. (Adapted from ref 381 IBI Oligonucleotide-directed RNase H sens1t1vity The S-100 extract was pre-incubated with RNAse H m the presence of no oligonucleotide (-oligo; lane 2), an oligonucleotide complementary to U2 snRNA (U2 L 15; lane 31. an oligonucleotide complementary to res1dues 64-79 of U3 snRNA (64-79; lane 4), or an oligonucleotide complementary to residues 80-96 of U3 (80-96; lane 5). All samples were then treated with DNase I followed by incubation with radiolabelled pre-rRNA processing substrate. Only the oligonucleotide targeted on residues 64- 79 of U3 snRNA resulted in RNase H digestion and loss of processing activity as shown by the absence of the (smaller) rANA product. Ne1ther the non-specific U2 oligonucleotide nor the U3 oligonucleotide targeted to the double-stranded reg1on of U3 snRNA (residues 80- 961 supported RNase H digest•on. (Adapted from ref 38).

to process the pre-rRNA. Oligonucleotides complementary 10 prominent singlestranded regions of the RNA are the most likely 10 be successful in directing digestion of this RNA in the presence of cellular components. For instance. of eight anti-U3 oligonucleotides tested, only one, corresponding to residues 64-79 of mouse U3 RNA, has been successful in promotmg RNase H directed remo\'al of U3 RNA activity from the mouse S-1 00 extract (38).

5.2.1 RNase H digestion of cellular RNAs in processing extracts The method is described m Protoco/15 and in Volume I, Chapter 4 (Section 6). Following RNase H treatment, assay an aliquot of the extract by Northern blot analysis to monitor the RNA digestion and test another ahquot for altered processing efficiency after incubation \\;th a 32 P-Iabelled pre-rRNA substrate. It is important to demonstrate that the loss of processing acti\ity occurs following 166

Cathleen Enright and Barbaro Sollner-Webb digestion of the RNA of interest, but not following parallel treatments with control oligonucleotides. It should al~o be remembered that an oligonucleotide may fortuitously allow destruction of an unanticipated RNA and that an apparent!~ negari' e result is hard to interpret unless one can be certain that the RNA destruction \\85 sufficiently complete to assure detection of an) potenual effect on processing.

Protocol 15. Oligonucleotide-directed RNase H digestion of RNA in the mouse S-1 00 processing extract" EqUipment and reagents • S · 100 processing extract and 0. 2 M D n stock solution, Protocol 6

e 5 x RNase H buffer 10.5 M KCI , 40% glycerol, 9 .76 mM DTI') • RNase free DNase IWorthmgton;

• Oligodeoxynbonucleotlde complementary to the target RNA sequence (1 1Jgi 1JI) • RNase H IBRL; 2 U/1JI)

2-5U'IIH • Equipment and reagents for studying processmg of pre·rRNA as described in Protocol 7

Method 1. Set up the followmg reaction mixture in a microcentrifuge tube on ice: • S-1 00 processing extract

10

~o~l

• oligonucleotide • 5 x ANase H buffer • RNase H

2 . Incubate the m1xture for 10 m1n at 37 °C, then for 20 min at 30 °C and then place it on ice. 3. Add 1 IJI of RNase -free DNase and incubate the mixture for 3 min at 37 °C followed by 7 min at 30 °C. 4 . Remove 6.4 IJI for Northern blot analysis of the targeted snRNA (see Volume I, Chapters 2 (Section 3.6) and 4 (Section 7 5)). 5 . Use the remainder of the reactiOn maxture from step 3 (9.6 jJI) to test the processing activity of the extract as descnbed 1n Protocol 7. For the processing assay, dilute the remainder of the reaction to 1 51JI with 10 fmol 32 P ·labelled pre-rRNA and A TP to yield a free magnesium 10n concentration of -0.5 mM. Although processing does not require ATP, including ATP 1n the reaction as opposed to adjusting the MgCI 2 to 0 5 mM results in sharper gel bands From ref. 38. Add DTI just before use from the OTT stock soluuon.

167

5: Ribosomal RNA processing in vertebrates Figure 68 shows the results of an experiment in which RNase H was used to demonstrate that U3 snRNA is required for 5' ETS processing in the mouse S-100 extract (38).

5.2.2 Oligonucleotide-directed RNase H digestion of RNAs in micro-injected oocytes The e111dogenous RNase H present in Xenopus laevis oocyres has been exploited to demonstrate the involvement of U3 snRNA on the processing of the cellular pre-rl~NA (16). In this case, the U3-specific oligonucleotide was injected tmo the oocyte nucleus and the endogenous RNase H performed the select e digest ion. It was concluded that the processing which occurs at the 5 ' -end of the 5 .8S rRNA region is dependent on the presence of U3 in the oocyte. Pr.( .. and Roeder. R. G. (1983). Vucleie Acids Res., II , 1475. Craig, N., Kass, S., and Sollner-\\'ebb, B. {1987). Proc. l\a/1 Aead. Set., USA., 84, 629. Kass, S. and Sollner-\\ebb, B. (1990) . •\lot Cell. Btol., 10, 4920. Bradford, 1\1. M. (1976). Anal. Biochem., 72, 248 Bartholomew, B .. Dahmus, M. E., and Meares, C. r. (1986). J. Bioi. Chem., 261, 14 226. Laemmli, U. K. (1970). Nature, 270, 680. Kass, S., Tyc. K.. Steitz, J. A .. and So liner-Webb, B {1990). Cell, 60, 897. Henderson, S., Ryan, K., and Sollner-Webb, B. (1989). Genes and De1· , 3, 212. Mougey. E B., Pape, L. K. and Sollner-\\ebb, B. (1993). Mol. Cell. Bioi.. 13 (In press). \1ougey, E. B.• O'Reilly, \1.. O~heim, Y., \tiller, 0. L.. Be}er. A. and Sollner\\ebb, B. (1993) Genes and De~·.. 7 (In pre~s)

171

Processing of transfer RNA precursors CHRIS L. GREER

1. Introduction 1.1 General methods T his chapter covers practical strategies and methods for studying the biochemistry of transfer RNA processing. Although processing of nuclearencoded tRNAs in yeast provides the primary focus for the chapter, essentially all of the strategies and many of the experimental methods apply equally well to the tRNAs of other organisms. This subject is generally amenable either to biochemical or genetic analyses. The methods in this chapter emphasize the biochemical approach and include the development of in vitro assay systems and characterization of reaction intermediates and products. Genetic methods for studying pre-tRNA substrate requirements are reviewed in ref. I and genetic approaches to the identification of trans-acting components are reviewed in ref. 2. Certain general methods applicable to tRNA processing, including fractionation of cellular RNAs, characterization of RNAs, a nd analysis of RNAprotein interactions, are described elsewhere in this book (Volume l , Chapters I, 2, and 5, respectively). The study of tRNA processing is, in many ways, unique within the RNA processing field. This assertion is based on both the unique physical and functional properties of tRNAs and the degree of complexity characteristic of tRNA biosynthetic pathways. Among the unique properties of transfer RNAs are their small size, stability, and relative intracellular abundance. These features make tRNAs attractive experimental subjects among cellular RNAs and the result has been the accumulation of a wealth of information regarding tRNA structure, function, and biosynthesis (see refs 3 and 4 for general reviews, and the compilation of tRNA and tDNA sequences in ref. 5).

1.2 tRNA structure and function The tRNAs function primarily in mRNA translation. Evolutionary constraints imposed by this crucial function are responsible in part for the high degree of conservation observed among tRNAs (reviewed in ref. 6). With the exception of certain mitochondrial examples, tRNAs share extensive common features

6: Processing of tra nsfer RNA precursors r.·

3'

E) - - Leader

/

e - -® Trailer

u Arm

D Arm

Ar m AC

Arm

Se quenc e Figure 1. Consensus pre·tRNA structure. The tRNA numbering scheme shown here is based on that descnbed in ref. 3. S1tes of variation in nucleotide sequence length occur in the 5' leader 3' trailer, and aotervening sequences. Invariant bases are represented by boxes lnd sem•·anvariant positions by ellipses. Positioos w1than the intervening sequeoce, S'leader end 3' tra1ler segments are Indicated by a colon, m•nus or plus sogn. respectJVely and are numbered from the adjacent exoo position. Solid hnes deoote segments of varyang length, N. The AA arm and AC arm mark the aminoacyl acceptor and anticodon arms, respectively. Arms commonly containing dihydrouridine and ribothymidine modif•cat1ons are marked D arm and T arm, respectively.

of primary, secondary, and tertiary structure. The primary sequence of mature tRNAs varies from 60-95 nt in length -with 13 imariaot and 12 semi-invariant position!>. E'\ten!>ive post-transcriptional modification is common with as many as 201t'o of the nucleotides of a mature tRNA species pre~ent in modified form (reviewed in refs 7, 8). A total of 52 different modifications have been characterized thus far, with some common to a majority of tRNAs and others unique to one or a few species (see the compilation in ref. 5). This facet of tRNA 174

Chris L. Greer structure greatly increase~ the number of di~tinct proces~ing reaction~ reqmred for the synthesis of any 'ingle tR "A species and also requires a rather large number of diverse processing pathways to produce the complete cellular repertoire of tRNAs. At the le,el of secondary structure. all of these tR'lAs form a consen·ed set of stem and loop structure which can be arranged 10 the traditional do,erleaf configuration. Thts structure and the corresponding nomenclature are summarized in Figure I. Significant length vanalion occurs only within the D-loop and variable arm segments \\ith the lengths of the remaining segments being highly consened. At the level of tertiary structure, aU tRNAs are thought to adopt an L-shaped configuration (reviewed in refs 9, 10). The small arm of the L-shaped ~tructure is formed by the aminoacyl acceptor (AA; Figure I) and T-stem helices, while the long arm consists of the D-stem and the anticodon stem and loop (AC; Fi~ure /). Tht~ conserved tertiary structure ts 'itabilized both by stad.ing interactiom between conserved helices and by tertiary interactions between conserved bases of the D- and T-loop segments, and the variable arm and D-stem regions. Thts high degree of consen·ation in both primary sequence· and threedimensional structure has two important implications for tRNA processtng. First, general processing enzymes may recognize their sub~tratte~ through conserved features within the mature, tRNA-like portion of the precursor, potentially at a distance from processing sites. Second, umque processing steps may requtre fine distinctions among tRNAs, potentially invoh ing discrete positive and negauve discnminators analogous to the determinants of tRNA identity in aminoacyl synthetase interactions ( II).

1.3 Overview of processing pathways The primary transcripts of tRNA genes m both prokaryote~ and eukaryotes must undergo a number of processing reactions to produce the mature, functional form of the molecule (12, 13). These reactions fall into three general categories: • removal of extraneous 'iequences • addition of non-encoded bases • introduction of modifications to the base and ribose moieties of specific nucleotides The temporal order of the~e processing events has been examined in a number of prokaryotic and eukaryotic systems. In all cases, there appears to be a preferred sequence of events for a given precursor since onl) a subset of the possible mtermediates has been detected. For example, processing in Xenopus oocytes is thought to start with trimming of the 5 '-end followed by maturauon of the 3 -end , splicing, and finally transport out of the nucleus (14). The addition of individual base modifications occurs at distinct stages in tills size-maturation process ( 15). While a similar order rna) apply to some precursors in yeast, others 175

6: Processing of transfer RNA precursors

may utilize different pathv\ays (16). Whether substrate specificity, functional compartmentation, kinetic constraints, or a combination of these facwrs define the processing pathways for individual tRNAs remain among the most pressing questions in tRNA bios)'"Tlthesis.

1.4 Biochemical analysis of tRNA processing The rest of this chapter is divided into three experimental sections intended to reflect the approximate temporal order one might follow in initiating a biochemical study of a tRNA processing reaction. Section 2 describes the preparation of tRNA precursors for use as substrates in processing reactions. Precursors may be purified from whole cells or be prepared by m vuro transcription. Section 3 includes the preparation of cell-free extracts active in transcription and specific processing reactions. Examples of the preparation 1 ~ complex nuclear extracts and purified splicing enzyme fractions are provided Section 4 describes the development of tRNA processing assays and provide\ examples of assays for measuring changes in the size and sequence of tht: precursor or for detecting the introduction of specific nucleotide modifications

2. Preparation of pre-tRNA processing substrates 2.1 Introduction A biochemical approach to the study of tRNA processing relics heavily on the provision of substrates of high purity and defined structure, and usually in large amounts or at high specific radioactivity. Additional requirements are the facility for manipulating substrate sequence or structure, the potential for site-speciftc labelling, and the ability to include or omit one or more of the specific processmg steps in preparing the substrate. Two general approaches are available fo r the preparation of processing substrates: • expression in vivo followed by isolation from whole cell RNA fraction • expression in vitro using homologous or heterologous transcription/ processu.g systems Each of these approaches has distinct advantages and limitations which should be considered when choosing an appropriate experimental strategy. These are described along with the methods themselves in the sections below

2.2 Preparation of nuclease-free carriers Labelled RNA substrates must often be concentrated from dilute solutions by precipitation with ethanol. At RNA concentrations less than 5 ~g mi. precipitation may be inefficient unless an appropriate carrier is added. For samples to be subjected to nuclease digestion (see Section 4 .3), the addition of a known amount of RNA carrier is appropriate. For samples to be used in processing or post-labelling reactions, glycogen should be added as carrier. Protocol/ describes the preparation of nuclease-free carriers for these purpose-. 1 76

Chris L. Greer Prot ocol 1 . Preparation of nuclease-free RNA and glycogen carriers

Reagents • 2. 0 M sodium acetate buffer pH 5.0 e TE buffer !1 0 mM Tns- HCI. pH 7 5. 1 mM EDTAl e 20'lb SDS

e s.O M NaOH e Proteonase K 120 U .mg protem, Sogma, p 65561

• Phenol chloroform : 1soamyl alcohol 125 24 : 11 • Sterile water; treat deoomzed dosulled water with 2 drops per htre of DEPC, autoclave 1 h, 120 °C, 16 p.s.i. • Either glycogen !oyster glycogen Sogma Type II G8751 l or unfractionated RNA from yeast ISigma Type VI R6625 o

Method 1. Dissolve the glycogen or RNA 1n TE buffer to 20-1 00 mg/ml final concentration. Note that this form of RNA must be carefully neutralized to d1ssolve it; add 5 M NaOH dropw1se with st1rnng. Use indicator strips to check that the solution is at pH 7. 5. 2. Add SDS to 0. 5%, proteinase K to 1 mg ml and incubate the solution for 30 min at 55 °C. 3. Extract the solution with 1 vol. of phenol. chloroform: isoamyl alcohol. Centrifuge at 2000 g for 5 min to separate phases. Repeat the extraction of the aqueous phase twice more and finally extract it with 1 vol. of ether. 4. Add 0. 1 vol. of 2 M sodium acetate buffer and 2 vol. of ethanol. Keep the mixture on crushed dry oce for 1 h to precipitate the RNA or glycogen.

5. Centrifuge for 15 min at 12 000 g, 4 °C. 6 . Air dry the pellet, resuspend it in 0.1 vol. of 2M sodium acetate buffer, and repeat the ethanol precipitation (steps 4 and 5) twice. 7 . Resuspend the dried pellet in sterile water to 20 mg/ml final concentration and store the sample at - 20 °C in 1 ml aliquots.

2 .3 Preparation of tRNA substrates from whole cells This approach is especially applicable where only limited information exists regarding the particular substrates or processing reactions to be studied and where an initial characterization i~ required. Substrates prepared in this way may have already undergone some processing steps, such as the introduction of base modifications, which might be required for the processing reaction to be studied. If so, analysis of the partially-processed species may provide insights into the nature of the naturally-occurring substrates. Disadvantages to this approach include the difficulties in obtaining highly purified fractions, the limited strategies available for specific radiolabelling of substrates either during in vivo synthesis or after their isolation, and the low levels of tRNA precursors normally present in ceUs. This Iauer difficulty may be partially overcome through the use of mutant cell lines or altered growth conditions \\Chich result in the 177

6: Processing of transfer RNA precursors accumulation of panially-processed transcripts. An example of this approach relies on the phenotype of the rnal-1 mutation in yeast (17): the accumulation of imron-comaining tR'\A precursors upon shift to the re:.tricti,·e temperature. This method (ba,ed on ref. 18) is de cribed here in t\\O part~: growth ana labelling ol the yeast cells (Protoco/2) and the extraction and i'olation of tRNA precursors (Protocol 3 ). The procedure relies on phenol e\.traction of nuclei I 00 IJM of the 182

Chris L. Greer initiating nucleotide (usually GTP). The low UTP concentration u-;ed in Protocol 4 reduces the reaction yield but results in high specific radioactivities

(- 5 x 103d.p.m./fmol).

Table 1 . Examples of phage promoter/tRNA gene fus1ons for the production of semisynthetic processmg substrates Applications

Promoter type/tRNA gene

References

Phage SP6 promoter

B

54

Schizosaccharomyces pombe tRNA 5 ••

A

55

Saccharomyces cerevisiae tRNALeu

A

56

Escherichia coli tRNAPhe

A

57

C,D, E

28,58

Saccharomyces cerev1s1ae tRNA Giu (mitochondrial)

Phage A promoter

Saccharomyces cerevisiae tRNA Tvr Phage T7 promoter

Saccharomyces cerevisiae tRNAPhe

D, E

59,60

Mycoplasma mycoides tRNAGiv

c

61

Schizosaccharomyces pombe tRNA5 ••

A

62

• uses made of the semisynthetic substrates 1n the references listed include: lA) formation of mature 5'-ends; (B) processing of 3'-ends; IC) addition of one or more specific nucleotide mod1f1cations; (D) presence of intron endonuclease and/or tRNA ligase activity; and (E) structural analysis.

Protocol 4 . In vitro tRNA gene transcription using T7 RNA polymerase Equipment and reagents • Phage T7 RNA polymerase 160 U/~1. Pharmacia 2 7-080 1-01 ) • Ribonucleotide stock solutions; separate 0. 1 M stock solutions in 10 mM TrisHC), pH 7.5 (ATP, 27-2056-01; CTP, 27-2066-01; GTP, 27-2076-0 1, UTP, 27-2086-01; Pharmacia) • [a·32P[UTP (10 mCi/ml, 3000 Ci/mmol; Amersham, PB. 10200-PB. 10203)

• Plasmid DNA containing the tRNA gene cloned next to a T7 phage promoter; prepared using QIAGEN cartridges (Oiagen 51 003) and the procedure recommended by the manufacturer • 5 x transcnpt1on buffer (0.2 M Tns-HCI, 50 mM MgCI 2 , 50 mM NaCI) • TE0.1 buffer (10 mM Tris-HCI, pH 7.5, 0.1 mM EDTA)

Continued

183

6: Processing of transfer RNA precursors

Protocol 4 . Continued • 50 mM OTT Store at - 20 °C for up to 1 week • Stop moxture 10.1 M EDTA, pH 8 0. 2% SDS. 2 mg lml proteinase K !Sigma, P

65561 • Phenol : chloroform: isoamyl alcohol

• Glycogen earner (20 mgtml) and sterile water prepared as 10 Protocol 1 • TBE buffer, 2.0 M ammonoum acetate, pH 7.0, gel sample buffer. and gel elutoon buffer as In Protocol 3 • Appropriate restriction enzyme and buffer

(25·24:11 • Polyacrylamode gel (15.5% acrylamode, 0 5% bosacrylamode 8 M urea, 1 >< TBE buffer 19 >e 22>e0.075 em); and electrophoresis equopment (see Protocol 31

• 5 x rNTP moxture 12.5 mM each of A TP GTP, and CTP plus 0 .05 mM UTPJ • • 0.4 M ammonoum acetate buffer. pH 7.0

Method A. Restriction of plasmid DNA template 1. Linearize the plasmid DNA by restriction digestion at the appropnate transcription ' run -off' site.

2. Extract the DNA by vortexing w1th an equal volume of phenol: chloroform . isoamyl alcohol. Centrifuge (2 min) in a microcentrifuge at room temperature to separate the phases.

3 . Precipitate the DNA from the upper (aqueous) phase by adding an equal volume of propan-2-ol and leaving the mixture at - 20 °C for 0.5 h. 4. Recover the DNA by centrifugation in a m1crocentrifuge for 15 mm. 5. Air dry the pellet, redissolve It on sterile water at 1 (JQI (JI and store it at -20 °C. B. In vitro transcription of DNA template

1 . Prepare a 50 (JI transcription reaction in a microcentrifuge tube on ice b r adding: • sterile H 2 0

12 IJI

• 5 x transcnption buffer

10 IJI

• on

10(JI

• 5 x rNTP mixture

10 (JI

• plasmid DNA template

11JI

e [a-32P] UTP

51JI

2 . Start the reaction by adding 2111 of T7 polymerase and 1ncubate for 1 h at 37 °C. 3 . Stop the reaction by the addition of 5 IJI of stop mixture and incubate for 15 min at 50 °C. 4. Add 40 IJI of 2.0 M ammonium acetate, pH 7 0, 1051JI sterile water 0.5 ml ethanol and keep the mixture on crushed dry 1ce for 1 h to precipitate the nucleic acids.

Continued 184

Chris L. Greer

Protocol 4 . Continued 5 . Centrifuge tn a microcentnfuge at 4 °C for 1 5 mm Wash the pellet once w ith 70% ethanol at 4 ° C . A ir dry the pellet and dissolve it in 5 ~I of sterile water. 6. Add 5 ~I of gel sample buffer and incubate for 5 mm at 65 ° C. 7 . Separate the RNA product by electrophoresis in the 15. 5 % polyacrylamide gel at 20 rnA until the bromophenol blue dye has migrated 17 em (2.0-2.5 h). Note that unincorporated nucleotides comigrate with bromophenol blue and that pre-tRNA transcnpts migrate behind the xylene cyanol marker Dtspose of the used electrode buffer as radioactive waste. 8 . Disassemble the electrophoresis apparatus leavtng the gel on the glass backmg plate. Wrap the gel and plate in cling film and mark the sides and corners with radioactive or fluorescent ink for later alignment of gel and film. 9 . V1suahze the RNA products by autoradiography (e.g. 5-15 min exposure using Hyperf1lm-MP and an intensifying screen) Develop the film according to the manufacturer's instructions. 10. Align the gel and film on a light box. W tth a razor blade, cut out the appropriate regton of the gel and elute the RNA from each gel slice by soaking it for 8 - 12 h at 37 °C in 2 ml of gel elution buffer as described in Protocol 3 (step 21 ). 1 1 . Add 0 .5ml of 2.0M ammonium acetate buffer, 2 . 5~1 of 20mg ml glycogen earner, 7 5 ml of ethanol, and prectpitate the RNA on crushed dry ice for 1 h. 12. Centnfuge for 20 mtn, 1 5 000 g at 4 °C. Dry the pellet and resuspend it in 0.35 ml of 0.4 M ammonium acetate buffer. 13 . Repeat the ethanol precipitation as in steps 9 and 10. 14. Wash the pellet in 70% ethanol. Resuspend the final dried pellet in TE0.1 buffer ( 10 000 c.p.m. ' ~I) and store at -20 ° C. • when labelling w1th a nucleoside tnphosphate other than [a- 32P] UTP, mod1fy the compos1t1on of th1s 5 x rNTP mtxture accordingly.

3. Preparation of tRNA processing extracts In addition to a method for preparing appropriate substrates, a biochemical approach to tRNA processing requires the ability to prepare active processing ext racts. Initially. the characterization of a processing reaction is often carried out with crude cell extracts. Long-term goals should include reconstitution of the reaction from highly purified components, obtained by fractionation of extracts from the original cell source or through the use of heterologous expression systems. The protocols described in this section represent examples from each of these three categories: preparation of a crude nuclear extract containing a variety of activities, partial purification of a processing enzyme from yeast, and the use of a bacterial expression system. 185

6: Processing of transfer R.\'A precursors Table 2. Sources of active tRNA processing extracts Activities

References

Escherichia coli

A,B,C

63-65

Bacillus subtilis

A

66

A. B

67

D

27

A,D

68

A

69

Saccharomyces cerevisiae

A B

54

Rat (liver)

A ,B

70

A

71

Extract source Eubacteria

Thermus thermophllus survey of diverse bacterial sources Archae bacteria Halobacterium halobtum Sulfolobus solfatancus Mi1ochondria

Human (Hela cells) Chloroplasts Euglena gracilts Spinach Ma1ze

A,B,C

72

A

72, 73

A ,B

74

A , B,C

31

Eukaryotes Saccharomyces cerevtsiae Schtzosaccharomyces pombe

A

55

Chlamydomonas

D

75

Wheat

A,B,C. D

A B

Drosophila melanogaster Bombyx mori

76

7~

79

A, B

80

Xenopus laevis

A,B,C,D

81-83

Human (Hela, KB. leukaemia cells)

A, B,C,D

84-88

Processmg steps exam1ned in the publications listed include: IAJ format1on of mature 5'-ends; fBI process1ng of 3'-ends; ICJ add1110n of one or more spec1fic nucleotide modifications. and (OJ presence of mtron endonuclease and ·or tRNA ligase activ1ty

186

Chris L. Greer

3.. 1 Preparation of crude cell extracts Crude or partially-purified extracts containing tRNA processing activities have been prepared from a wide variety of sources and the literature provides an excellent starting point in designing an experimental strategy. Table 2 provides an over,iew of this literature with examples from eubacterial, archaebacterial, eukaryotic nuclear, and eukaryoric organellar sources. Many of these extracts are active for transcription as well as certain processing reactions allowing their use for studying coupled synthesis and maturation reactions. Advantages and disadvantages in the use of these extracts are both rooted in their complexity. Thus, one advantage is the ability to examine processing steps requiring several components or multiple processing reactions. A clear disadvantage is the presence of competing activities, including nucleases, proteases, phosphatases, etc. H owever, with appropriate regard for the inherent limitations, these complex extracts can be used effectively to obtain useful information regarding specific tRNA processing reactions. Protoco/5 describes the preparation of a crude yeast nuclear-extract fraction and is adapted from the procedure described in ref. 29.

Protocol 5. Preparation of a nuclear extract from yeast Equipment and reagents • Oounce homogenizer (40 ml size) woth large (A) and small clearance (8) pestles IKontes, 885300-00401 • Oak Ridge type plastic centrifuge tubes (30 ml, capped I

• Saccharomyces cerevisiae straon JHRY20-2CO 1 (protease-deftcient•, MATa, his30200, ura3-52 . /eu2-3. 112, pep4-0 1 .. URA3; other proteasedefocient straons can be substotuted (e.g. 208- 12. Amerocan Type Culture Collection, 206261

• 1% SOS • Sl and Sll spheroplastlng buffers 1100 ml each). Prepare 200 ml of 1.0 M sorbitol, 10 mM MgCI2 , 50 mM Tris-HCI, pH 7 .8. Oivode thos into 2 ahquots of 100 mi. Just pnor to use add 3 ml of 1 0 M OTT to one aliquot to make Sl buffer and 0 .3 ml of 1.0 M OTT to the other aliquot to make Sll buffer • Sill lysos buffer (1 00 ml; 15 mM KCI , 5 mM MgCI 2 , 0 .1 mM EOTA. 10 mM Hepes - KOH pH 7 81

• 1.0 M OTI ( 10 ml); make up fresh using sterile water (see Protocol II

• Oialysos tubong and doalysos buffer (3 litres; 0 . 1 M KCI, 10% glycerol, 0 . 1 mM EOTA. 20 mM Hepes-KOH, pH 7.81. Autoclave (1 h, 120 ° C, 16p.s.i.l; add 3 ml of 1 0 M OTT JUSt before use

e YPD medium (Protocol 2 )

• Solid INH.12SO. and 4 .0 M INH 12SO.

• Yeast lytoc enzyme (1 0~ U 'g, ICN 8iomedocals 190- 123)

Method NB Carry out steps 7-3 at room temperature and all other steps at 2-4 °C.

1. Grow the yeast cells in 1 litre of YPD medium at 30 ° C with aeration until A 600 =6.0- 10.0. Harvest the cells by centrifugmg at 3500g for 5min.

Contmued

187

6: Processing of transfer RNA precursors

Protocol 5. Continued 2. Resuspend the cells m 33 ml of Sl buffer and d1v1de the suspens1on into two Oak Ridge-type cemtrifuge tubes. Centrifuge at 3500 g for 5 min 3. Resuspend each pellet in 16.5 ml of Sll buffer and add 1.6 mg of yeast lytiC enzyme to each tube. Incubate the cells at 30 °C with gentle agitation until spheroplasting is complete. Momtor the spheroplasting as follows. Before addition of the lytic enzyme and at 10 min intervals thereafter, remove a 10 JJI sample of cell suspension and add it to 2 ml of 1% SDS. After 2 min, measure the A 600 . Spheroplasting is complete when the absorbance falls to 20% of the time zero value (usually after 30-70 mini. Excessive or incomplete spheroplasting greatly affects the final activity of the extract. 4. Collect the spheroplasts by centrifugmg at 2000 g for 5 m1n at 4 °C Resuspend them gent I•( with a glass rod m Sll buffer ( 16.5 ml per tube I and repeat the centrifugation 5. Resuspend each pellet m 10. 5 ml of Sill lysis buffer. Combine the contents of the two tubes into the Dounce homogenizer (prechilled) and homogenize once gently with the A pestle. Incubate the suspension for 20 min on ice. 6. Lyse the spheroplasts bv homogenizing VIgorously 3 t1mes w1th the B pestle. 7 . Collect the particulate fract1on (includmg the nuclei) by centrifuging at 17 000 g. 10 min at 4 °C and carefully pour off the supernatant. 8. Resuspend the pellet in 8 ml of Sill buffer, transfer the suspension t o the homogenizer, and use an additional 2 ml of Sill buffer to rinse out the centrifuge tube. Homogenize the comb1ned suspension once gently w1th the A pestle. 9. Calculate the volume of the 'breakage pellet' (for use in step 11 below) by measuring the final volume of homogenate m step 8 and subtracting 10 ml (the total volume of buffer added). Add 0 .2 vol. of 4 .0 M (NH 4 ) 2 S0 4 . Extract fo1• 10 min on ice with occasional mixing.

10. Remove particulate material by centrifuging at 1 50 000 g for 1 hat 4 °C Remove the supernatant carefully and measure the volume. Add 0 .25 g/ml of solid (NH4 bS04 . Mix gently and mcubate for 10 m1n on ice

11 . Collect the precipitate by centrifuging at 17 000 g for 30 min at 4 °C Resuspend the pellet .n 0 . 5 times the volume of the ' breakage pellet (calculated in step 9).

12. Dialyse the suspension against one litre batches of dialysis buffer (making 3 changes of buffer at 1 h intervals) at 4 °C with stirring.

13. Transfer 50 Ill aliquots of the d1alysed extract to m1crocentrifuge tubes and store them frozen at - 70 °C. • See ref 30.

Typical yields from this procedure are 2-3 ml of an extract containing 8-12 rng total protein per millilitre. This extract is effic1ent in tRNA gene transcripuon (29) and is active in a number of processing reactions including 5 ' -end maturation, 3 ' -end trimming and CCA addition, and base modifications su ... ll as the formation of pseudouridine, dihydroundine, ribothymidine, and certa n 188

Chris L. Greer methylated derivatives (when supplemented with 0.1-l mM S-adenosylmethionine). The extract also contains a small amount of tRNA endonuclease and RNA ligase, but splicing IS inhibited at the salt concentrations typically used in transcription reactions (30). Thus end-mature, unspliced precursors accumulate in reactions using intron-containing tRNA gene templates. Accumulation of 5' and 3 end-extended precursors can be accomplished by carrying out transcription reactions at a reduced temperature (12-18 °C).

3.2 Partial purification of processing activities Deterntination of the reaction kinetics of RNA processing, the structures of proce sing intermediates and products, and the concentration dependence of substrate, enzyme, or co-factors is often not feasible in crude extracts due to the presence of competing activities and the poorly defined enzyme, co-factor, and RNA content of the extract. Although reconstitution from pure components may be the ideal way to investigate these aspects, the use of partially purified fractions provides a realistic intermediate step in the characterization of a processing system. Purification procedures are necessarily idiosyncratic and are developed empirically. However, a common strategy is the initial application of conventional protein fractionation techniques which rely on the general physical properties of the proteins of interest, followed by specific affinity methods which take advantage of distinct functional properties. Conventional techniques often applied to tRNA processing activities include differential ammonium sulphate precipitation, ion-exchange chromatography (particularly using heparin-agarose, phosphocellulose, DEAE-cellulose, hydroxyapatite, and certain FPLC resins, including Mono Q and Mono S from Pharmacia), and molecular sieving columns. Affinity chromatography methods include the use of immobilized tRNA or synthetic RNA resins, and tRNA-mediated elution from conventional resins (32, 33). Protocol 6 describes a partial purification of yeast tRNA endonuclease. A more extensive purification procedure can be found in ref. 34. The method described here leads on from the preparation of the nuclear extract described in Protoco/5 which should be consulted before proceeding. This method takes advantage of the observation that yeast endonuclease behaves as an integral membrane protein and is adapted, in part, from the method developed by Peebles eta/. (35). Note that in some experimental systems, such as Xenopus oocytes (36), the endonuclease may be found in the soluble fraction and so this purification procedure should be modified accordingly.

Protocol 6. Preparation of partially-purified endonuclease from yeast

Reagents • Yeast nuclear pellet prepared as described 1n Prococol 5, steps 1-7

• 1.0 M OTT (see Prococol 5l

189

Contmued

6: Processing of transfer RNA precursors Protocol 6 . Contmued • 20% lv •v) aqueous Triton X- 100 IBio Rad . 161 ·0407); equilibrated overnight prior to use • 0. 15 M PMSF (Sigma P 7626). prepared fresh in propan-2-ol and added fresh to buffers just pnor to use; NB Thts protease mhibitor ts roxie and should be handled With care. • Hepann-agarose (0 8- 1 mg hepann ml bed volume; B1o-Rad 153-61731 Two columns are requ1red. one containing 10 ml bed volume per litre of starting cell culture (see Protocol 5 I and one containIng 1 ml bed volume (in a disposable column or syringe) each equilibrated 1n ME buffer

• HSM buffer ( 1.0 M KCI, 0 . 1 M Tns- HCI pH 8.0, 10% glycerol, 2 mM EDTA. 5 mM spermid1ne, 1 mM DTI ', 1 mM PMSF •t • LDM buffer (20 mM Tns - HCI, pH B 0, 10% glycerol. 5 mM 2-mercaptoetha.nol 1 mM EDTA. 0 . 2 mM PMSF', 0 .02 % Triton X- 100) • ME buffer (LDM buffer but With o _g q Tnton X - 100 and 0. 1 M (NH,t 2SO,,) • ME buffer w1th 2% Tnton X -100, 0.1 M (NH.t 2SO. • Storage buffer A (20 mM Tris- HCI, pH 8 .0 . 0 2 mM EDTA. 0 . 5 mM DTI 0 . 5% Tnton X -100, 35 % glycerol)

• 0.5 M sperm1d1ne (phosphate salt, Sogma, S 0381). pH to 7. 5 With HCI; store frozen at - 20 °C

Method NB Carry out all the following procedures at 2-4 °C. 1. H1gh-salt wash ofnuclei. Resuspend the yeast nuclear pellet in 7 5 ml of HSM buffer. Collect the nucle1 by centrifugation at 1 7 000 g for 1 5 min at ..:~ °C 2. Low-detergent wash . Resuspend the pellet in 7 .5 ml of LDM buffer and incubate the suspension for 10 min on ice. Recover the membrane fraction by centrifuging at 1 50 000 g for 60 min at 4 °C. 3. Membrane extraction. Resuspend the membrane pellet in 10 ml of ME buffer containing 2% Triton X-1 00. Incubate the membranes for 30 m11'1 on ice with occasional miXing. Remove any remain1ng particulate material by centrifuging at 1 50 000 g for 60 min at 4 °C.

4. Apply the supernatant to the large heparin-agarose column. Collect 2 ml fractions throughout each of the followmg wash and elution procedures Wash the column w1th 20 ml of ME buffer

5. Elute the column stepw1se w1th 20 ml of ME buffer conta1mng 0.25 M (NHl4bS04, and then 20 ml of ME buffer plus 0.45 M (NH 4 ) 2 S0 4 • The endonuclease elutes in the latter fractions. 6. Dialyse the eluted endonuclease against 3 x 1 litre of fraction buffer for 1 h each t1me at 4 °C with stirring. 7 . Concentrate the dialysed endonuclease by applying 1t to the 1 ml hepann -agarose column. Elute the endonuclease with 2 ml of ME buffer plus 0 .45 M INH 4 l2S04. 8. Dialyse the eluate overmght at 4 ° C agamst one litre of storage buffer. Store the dialysed endonuclease solution at - 20 °C 1n 0 . 1 - 0 5 ml aliquots. • Add these reagents JUSt prior to use from stock solutions.

190

J

Chris L. Greer This procedure yields an endonuclea~e preparation which is approximately equivalent to Fraction V of ref. 35. Endonuclease activity averages 100 U/ ml where I Unit of endonuclease cleaves 20 pmol pre-tRNA substrate min t under standard reaction conditions (35). Significantly, the preparation contains only traces of contaminaung phosphatase and nuclease activities and is suitable for most studies of processing reactions including thetr substrate requirement'>. reaction products, and general reaction kinettics.

3.3 Preparation of highly-purified processing components Ideally, comprehensive biochemical characterization of a tRNA processing step should be carried om through its reconstitution from purified components. Often, thb is accomplished by extensive purification of processing enzymes from homologous cell extracts. This approach is particularly applicable to reactions requiring multiple components or complex assemblies, does not include a requirement for cloned genes or cDNAs, and allows for the inclusion of essential post-translational modifications. However, this approach can be experimentally challenging or even impracticable when the c•ell or tissue source is difficult to obtain in large quantities, or when the processing components are present at only trace levels in cells or are unstable in homologous extracts. An alternative approach is expression at high levels in a heterologous system followed by purification by conventional or .affinity methods. A prerequisite for this approach is an appropriate cloned DNA sequence encoding the desired product. Advantages include the ability to produce altered processing components in vitro by modifying the expressed gene (particularly important where the gene encodes an essential product) and the beneficial effect of efficient and high-level expression on both the degree of purification required to obtain a homogeneous product and to produce large quantities of the purified product. Disadvantages in this approach are those that favour homologous extracts: limited post-translational processing of the expressed protein factor and difficulty in reconstituting multicomponent or complex systems. Prorocol 7 provides an example of a bacterial expression system which allows the rapid and efficient punfication of .a yeast processing enzyme (RNA ligase). The bacterial expression vector used in this procedure, pDAKC (37), allows expression of yeast ligase as a translational fusion protein with bacterial dihydrofolate reductase (DHFR). This vector, derived from plasmid pKK223-3 (38), includes both the rae promoter for high-level expression on induction with isopropyl-13-D-thiogalactos.ide (IPTG), and a transcription terminator, rrnB T l/T2. The fusion protein, can be purified from bacterial cell extracts using a rapid and simple affinity method based on DHFR binding to metbotrexate-agarose (39). The affinity-purified protein can be used directly in processing studies, or contaminating nucleic acids and enzyme-bound folate can be removed using a DEAE-cellulose flow-through procedure as described in Protocol 7. 191

6: Processing of transfer RNA precursors

Protocol 7 . Purification of DHFR-Iigase fusion protein from E. co/, Equipment and reagents 47. 5mM K 2 HP04 1. 0 18M KCl, 10'1' glycerol)

• Dosposable chromatography columns (QS-Y, lsolabl

• E. coli, strain SW 1061 (pDAKC). C600 Dlon 100, hsdR , hsdM· , supE, thr- 1, leuB6, thi- 1, lacY1, IF' laci50 :: TN5); transformed woth the ligase expressoon vector, pDAKC

• M6E buffer (0. 1 M potassoum borate, pH 9.0, 1.0 M KCl, 1 mM EDTA, 2 mM folic acod, 1 mM 2-mercaptoethanoll. A del the folate and mercaptoethanol just prim to use

• LKA growth medium (8 gil NaCI, 5 gil yeast extract, 10 g/1 Bacto-tryptone (Dileo Laboratories) plus 50 "g ml each of kanamycin and ampicillin)

• MB buffer (50 mM potass1um phos phate, pH 6 .0 h.e. 44 mM KH 2 PO,. 6 mM K 2 HP0 4 ), 0 . 2 M KCl , 0 . 5 mt.-1 EDTA, 1 mM 2-mercaptoethanoll

• Lysozyme stock solution 117 mgl ml in 10 mM Tris-HCl, pH 8.0; Sigma L 6876} • Brij 58 solution (polyoxyethylene 20 cetyl ether; 50 mglml aqueous solution; Sogma P 58841 • DNase 1 solutoon 113 mg ml stock 1n 10 mM Tris-HCl, pH 8.0 ; S1gma DN-251 e 0.1 M lPTG • 0 .2 M PMSF (P 7626 Sigma), prepared fresh on propan-2-ol and added fresh to buffers just prior to use. NB Th1s protease inhibiror is toxic and should be handled with care • 0. 5 M EDTA (pH 8 .0 woth NaOHl

• Methotrexate-agarose. Equilibrate moetho trexate - agarose slurry IPoerce 20206 H in MB buffer Use 20 ml of slurry, contaon· ing 10 ml (settled volume) of moetho trexate - agarose and 10 ml of MB buffeo e DEAE -cellulose IWhatman DE-5 2. 4057-200). Equilibrate 3 ml lsnttled volume) per litre of startong cell cult•Jre in DEAE buffer • MW buffer 1200 mM potassium phO$ phate, pH 6.0 (i.e. 176 mM KH 2PO. 24 mM K 2HP0 4 1. 1 M KCl, 1 mM 2-mercaptoethanoll • FR buffer (100 mM potassium phosphate pH 6.0 ) (i.e. 5 mM KH 2P04 and 95 mM K 2HP04 l

• 1.0 M MgC12 e 40 mM Tros-HCl. pH 8.0 • Solid 1NH4 l 2 S0 4 , finely ground e DEAE buffer (50 mM potassium phosphate pH 8.0 (i.e. 2. 5 mM KH 2PO• •

• Storage buffer B ISO mM potassium phosphate, pH 6.0, 0 .1 M KCI, 5 mM 2-mercaptoethanol, 35% glycerol)

Method NB Steps 4-13 are carried out at 2 - 4 °C with prechilled equipment and buffers

Growth of bacterial cells 1. Inoculate 500 ml of LKA growth medium with bacteria and grow overnight at 37 °C with aeration until the A 600 is approximately 3 .0 . 2. Inoculate 10 litres of LKA medium with 500 ml of the overnight culture and grow at 37 °C until the A 600 is approximately 1.0 (3.5-4.0 h). 3 . Add IPTG to 0 .5 mM and continue the growth for 45 mon (A 600 should reach 1.2- 1.6).

CoMinued 192

Chris L. Greer Protocol 7. Continuc<Jd 4 . Harvest the cells by centrifugcng at 4200 g for 10 men at 4 °C Resuspend the cell pellet en 500 ml of 40 mM Tris-HCI, pH 8.0, and centrifuge at 6000 g for 1 5 min at 4 °C en a preweighed centrifuge tube. Record the weight of the cell pellet (typccally 1 8-2.8 g per litre of original culture). Store the cells at - 70 °C, they are more readily lysed (in step 6 below) after a freeze/thaw cycle. Extraction of fusion protein

5 . Add 2.14 ml of 40 mM Tris- HCI, pH 8.0, per gram of cell paste and measure the volume IV, I of the resuspended cells. Add 0.005 x V, of 0.2 M PMSF, 0.01 x V, of 0.5 M EDTA and 0.04 x V, of lysozyme stock solutcon. IncubatE! the mixture at 2-4 °C for 30 min with slow stirring. 6 . Add 0.04 x V, of Brij 58 solution. Incubate for 30 min at 2- 4 °C with slow stirnng The! viscoscty should increase wcth cell lysis 7. Add 0 082 x V, of 1 0 M MgCI2 , and 0.004 x V, of DNase I solution. and incubate for 30 rnen Highly viscous samples may require the additcon of a second aliquot of DNase I solution and further incubation for 30 men. 8 . Remove the cell debris by centnfugeng at 23 000 g for 1 h at 4 °C Carefully recover the supernatant and measure its volume. Add 0.226 g of solid (NH 4 ) 2S0 4 per ml of supernatant; make the additions in small amounts wactmg until the (NH 4 ),S0 4 has fully dissolved before further additcon 9 . Centrifuge the suspension at 12 000 g for 40 men at 4 °C. Recover the supernatant and measure cts volume. Add a further 0 212 g of solid (NH 4 ),S04 per ml of supernatant slowly with mcxcng. Centnfuge at 1 2 000 g for 40 rnin. 10. Resuspend the protein pellet in 18-20 ml of MB buffer. Measure the protem concentration by the method of Bradford (40). Adjust the final concentration to 20 mg protein/ml wcth MB buffer (typical yields are -75 mg protein 10er litre starting culture). 11 . Add 1 ml of methotrexate-agarose slurry per litre of starting culture. Place the resulting suspension in a dialysis bag and dialyse it agacnst two 4 litre batches of M B buffer for 1 2-1 6 h (mix the suspension occasionally by cnverting the dialysis bag). 12. Mcx the dcalysed sample thoroughly and transfer ct to a dcsposable chromatography column, allowcng the column to flow at -0. 5 ml/mcn. Collect 7. 5 ml fractions. 13. Wash the column at 0. 75 mi, men wcth MW buffer untcl the eluate reaches A 280 of that of the peak fraction. Dcalyse tthe pooled fractions against 3 changes of FR buffer ( 1 litre each time) over 10-12 h.

Continued

193

6 Processing of transfer RNA precursors Protocol 7. Continued 15. Concentrate the eluted protein by prec1p1tation w1th (NH4 12SO, "S follows: measure the volume of the pooled fraction and add 0.516 g INH4 12 S0 4 per ml Collect the precipitate by centrifugmg at 12 000 g ' or 45 mm.

16. Resuspend the pellet in 5 ml of OEAE buffer and d1alyse the solut1on against 3 changes of this same buffer ( 1 litre each time over 12-15 hi.

17. Apply the dialysed solut1on to the DEAE-cellulose column. Maintain the flow rate at 1.5 ml min and collect 2 ml fractions. Wash the column With 1 column volume of DEAE buffer. collecting fractions as before. Measure the prote1n concentrations of the fractions and pool those fractions w ith values ~ 10% of the peak fraction

18. Concentrate the purified protem by precipitation With (NH 4 hS0 4 as described in step 15. 19. Resuspend the prec1p1tated protem in 1 ml of storage buffer Band dialyse 1t against 3 changes of this same buffer, 1 litre each time over 15-20 h. 20. Store the dialysed preparation 1n aliquots at -20 °C.

L_

Final preparations obtained using Protocol 7 typically contain 1-2 mg protein/ ml, corresponding to yields of 0.1-0.2 mg per litre of starting cell culture. An example of the purification of the DHFR/ Iigase fusion protein is shown in Figure 2, including the methotrexate-agarose affinity chromatography (Ftgure 2A) and SDS-PAGE analysis of pooled fractions obtained by affinity chromatography (Figure 2B). Ligase activity in the final purified fraction is typically I000-3000 U/ml, where I Unit of enzyme activity ligates I pmol tRNA substrate per min 1 (41). Other important features of the procedure include the following: (a) The peptide linker in the fusion protein includes a Factor X cleavage me which allows separation of the yeast and bacterial protein segments by endoproteinase treatment (42). However, the author has found that the acti,ity of the ligase is not affected by the presence of the DHFR segment. (b) Induction of the cell cultures with lPTG for long periods ( ~ 2 h) resu ts in the accumulation of subfragments rather than full-length fusion protein. In fact, for many deletion derivatives, recovery of the intact fusion protein is better wtth uninduced cells. Thus, a comparison of induced and uninduced cell extracts should be made before settling on a final protocol. (c) The ionic strength of the DEAE buffer in the DEAE chromatograph"' is intended to aJiow retention of contaminating folate and polynucleotides without retention of the fusion protein. Recovery in this step is sensith·e to smaJI changes in ionic strength and so the conductivity of buffer, eluant, and ~ample should be carefully monitored. 194

Chris L. Greer

A.

B. ,..

0.8

rrc;110ns Doofed

-

-
<splicing cocktail) to maintain enzyme solubility. 196

Chris L. Greer (b) The stabiJizing effect of spermidine varies among pre-tRNA subtrates wit h a modest effect for some and a strict requirement for added spermidine for others (cf. ref. 35).

(c) Joining by RNA ligase involves two NTP-dependent reactions; a GTPdependem substrate phosphorylation and an ATP-dependent adenylylation of protein and RNA intermediates (Belford, H. G., Westaway, S. K., Abelson, J. , and Greer, C. L., in preparation). The nucleotide mixture in the assay provides both NTPs at concentrations 2-5 times their respective K app (NTP concentration required for half-maximal velocity) values. (d) The splicing reaction is optimal over a relatively narrow range of NaCl and KCl concentrations (5-50 mM). However, glutamate, a more relevant physiological anion than Cl - (45), can be used at con cent rat ions up to 0.5 M with little deleterious effect on splicing and at the same time reducing the non-specific binding of ligase by RNA (46). Thus, as has been suggested for other protein/ nucleic acid interactions, the use of glutamate as a coumerion may, under certain conditions, enhance specificity. (e) Digestion of protein components with proteinase K (in the stop mixture, see Protoco/8, step 3) prior to precipitation with ethanol improv•es recovery and enhances subsequent electrophoretic resolution of the reaction products. This is especially apparent for reactions containing concentrated or complex protein mixtures.

Protocol 8 . Pre-tRNA splicing assay Equipment and reagents •

32

P-Iabelled pre-tRNA substrate

I- 5000 d.p.m.lfmol; 5 fmoi/IJII prepared as in Protocol 3 or 4 ; each assay requires 5 fmol

• Dilution buffer (2 5 mM NaCI, 20 mM Hepes-KOH, pH 8 .0, 1 mM OTT O.SmM EOTA, 0 . 5% Triton X- 100, 20% glyceroll

• Yeast endonuclease prepared as in Protocol 6 ; each assay requires 0.1-1 .0 x 10 • units • OHFR-Iigase prepared as in Protocol 7; each assay requires 0.1-1 0 x 1o- 3 units • 1M

on (see Protocol 5 I

• 0 .25 M spermidine 1Protocol6), pH 7. 5 with HCI • Sterile water (Protocol 11

• to x

• 10 x nucleotide mixture ( 1 mM A TP and 0. 1 mM GTP m TEO 1 buffer) ; store at - 20 ° C

splictng cocktail 10.25 M NaCI, 0 .2 M Hepes - KOH, pH 7.5, 50 mM MgCI 2 , 25 mM spermidine, 10 mM on. 4 % Triton X- 100)

• Polyacrylamide gel I 12% acrylam1de, 0.4% bisacrylamide, 8 M urea. 1 xT8E buffer; 20 x 20 x 0. 1 em); see Protocol 3 • Gel sample buffers. elution buffer, 2.0 M ammonium acetate, pH 7.0 . TBE buffer, Quik-Sep columns as in Protocol 3. • Stop mixture and TE0. 1 buffer as in Protocol 4 • 2.0 M sodium acetate, pH 5.0

CorJtinued 197

6: Processing of transfer RNA precursors Protocol 8 . Continued

Method 1.

Dilute enzyme stock solutions, where necessary, using dilution buffer and leave to equilibrate for 0 . 5-1 h on ice (equilibration after dilution is essential for detergent-solubilized fractions) . Dilute endonuclease fractions to 0. 1-1 x 10 3 Units/IJI. Dilute ligase fractions to 0. 1-1 x 10 2 Unitsl i.JI.

2.

To a microcentrifuge tube on ice, add the following in the order given.

3.

• sterile water • 10 x splicing cocktail • 10 x nucleotide mixture

5 IJI 1 IJI 1 i.JI

• yeast endonuclease eDHFR-Iigase • 3 2P-Iabelled pre-tRNA substrate

1 i.JI 11JI 1 i.JI

Incubate the mixture at 30 °C for 5-20 min. Stop the reaction by adding 0.1 vol. of stop mixture. Incubate for 10 min at 50 °C.

4 .(a) For procedures requiring fine electrophoretic resolution (i.e. molecules differing by less than 1 Oo/o in chain length) Concentrate the reaction samples prior to electrophoresis by ethanol precipitation: adjust the volume to 80 i.JI with sterile water, add 20 l.ll of 2M sodium acetate, pH 5.0, 20 i.Jg of glycogen carrier (Protocol 1 ), and 0.25 ml of ethanol. Leave on crushed dry 1ce for 30 min. Recover the precipitated RNA by centrifugation at 12 000 g for 20 m1n at 4 ° C. Resuspend the dried pellet in 5 i.JI of gel sample buffer. (b) For procedures requiring limited resolution (i.e. molecules differing by more than 10% in chain length).

5.

Analyse the RNA products directly by electrophoresis by adding 1 assay volume ( 10 i.JI) of gel sample buffer. Denature the samples immediately prior to electrophoresis by incubating for 5 min at 65 °C.

6.

Load the samples onto the polyacrylamide and electrophorese as described in Protocol4 (step 7) allowing the bromophenol blue marker to reach the bottom of the gel (2.5-3 hat 20 mA) .

7.

Disassemble the electrophoresis apparatus leavmg the gel on the glass backing plate. Wrap the gel and plate in cling film and mark the sides and corners with radioactive or fluorescent ink for later alignment of gel and film .

8.

Visualize the RNA bands by autoradiography (e.g. 8- 12 h exposure at - 70 °C using Hyperfilm-MP and an intensifying screen). Develop the X-ray film according to the manufacturer's instructions. Align the gel and the autoradiograph on a light box. With a razor blade, cut out slices from the gel corresponding to the tRNA precursor and its reaction products and determine the amount of radioactivity present by measuring the Cerenkov radiation . Recover the RNA products from the gel slices by elution as described 1n Protocol 3 (steps 21-24).

9.

10.

198

1 0 ' - - - - ' - - - ' - - _ _ . J - - - ' - - - - ' ---' 3 4 5 0 2 [ GTP]

0I

05

1

25

5

(GTP)

(uM)

Figure 3 . Pre·tRNA splic1ng assay. (A) Assays (10 iJI, see Protoco/ 8) contained pre·tRNA~'~­ substrate, 10 iJM ATP, and the indicated concentration (iJM) of GTP Reactions were incubated w1th endonuclease for 10 min at 30 °C followed by the addition of ligase and further incubation for 20 min at 30 °C The products were separated by gel electrophoresis and visualized by autoradiography (Protoco/8, steps 7 and 8). The identities of labelled products and the posit1on of the xylene cyanol marker are indicated on the left as follows: pre-tRNA, pre-tRNA substrate; tRNA, spliced tRNA product; halves. exon products of cleavage by tRNA endonuclease . (B) The reaction products separated by gel electrophoresis in panel A were quantified by measuring the Cerenkov radiation in gel slices (see Protoco/8, steps 9 and 10). L1gase activity (o/o Joined) was calculated as described in ref 44

The results of a series of splicing assays using labelled pre-tRNA~' as substrate are shown in Figure 3. Panel A shows the autoradiograph of the gel. The endonuclease cleavage products (tRNA 5' and 3' exons and the intervening sequence) migrate between the dye markers in this gel system. The joined product (spliced tRNA) migrates behind the xylene cyanol marker and is well resolved from endonuclease products and from residual precursor.

4.3 Assay of base modifications and tRNA splice junctions 4.3.1 Strategy Transfer RNA biosynthesis includes the post-transcriptional introduction of a complex and extensive set of specific base and ribose modifications (reviewed 199

6: Processing of transfer RNA precursors in refs 7, 8). These modifications are essential for normal t RNA function. influencing decoding potential, translational efficiency and fidelity, and conformational stability. Reactions required to generate the full range of modifications found in a mature tRNA include simple methylation, reduct1on and isomerization reactions, as well as more complex side-chain addition and transglycosylation reactions. These reactions are catalysed by an extensive set of site-specific modifying enzymes which are thought to act at discrete stages in a tRNA processing pathway (15). The functions of these specific modifications, the substrate recognition properties and catalytic mechanisms of the modifying enzymes, and the ordering of the processing events are important areas of study in tRNA biosynthesis. Most current biochemical methods for anaiysing specific tRNA modification reactions involve the following steps: (a) Incubation of pre-tRNA substrates with proctessing extracts; (b) Isolation of the reaction products; (c) Digestion of the RNA product to its constituent nucleotides, or nucleosides; and

oligonucleotide~

(d) Separation of the modified constituents from their unmodified counterparr.. on the basis of distinct chemical or physical properties. The requirements for the first step have been discussed in Sections 2 and 3 Isolation of the reaction products may simply consist of phenol extraction followed by ethanol precipitation (see Protocol3) or more extensive purification by gel electrophoresis (see Protocol 8) or chromatography (22, 23). Isolated reaction products are then digested to their constituent oligonucleotides. nucleotides, or nucleosides by treatment with the appropriate nucleases or chemical cleavage reagents. In this step, the effe,;ts of modifications on the cleavage reaction must be considered and examples of the use of nuclease PI and snake venom phosphodiesterase are described in Section 4.3 .2. In the final step, digestion products are resolved by TLC (8, 47), HPLC (48, 49). or combined liquid chromatography/ mass spectroscopy (LC/ MS; 50, 51). The digestion products may be detected and quantified by UV absorbance when unlabelled substrates have been used or by radiometry or autoradiography for the products of labelled substrates. Labelling with ! 32P] orthophosphate m vivo (Protocol 3) and post-labelling procedures in which the RNA is labelled after synthesis (47, 52, 53) result in uniform labelling and potentially allo\\ the detection of all the modified nucleotides present in a single digestion Where the labelled RNA substrate has been synthesized in vitro, the ability 10 detect a specific modified nucleotide depends both on the choice of the labelled nucleotide and the specificity of the cleavage reagent used in the subsequent digestion (i.e. whether phosphates are retai ned in the 5' or 3' position in cleavage products). These two factors must be considered in designing procedures which include in vitro transcription. For example, transcription with 200

Chris L. Greer [ a-32 P) GTP and subsequent digestion with nuclease P l (to generate 5 mononucleotides) would be effective in detecting modifications at G residues but would not allow the detection of other modified bases. The tRNA products of the yeast splicing enzymes have a characteristic junction structure consisting of a 2 ' -phosphomonoester-3 ,5 ' -phosphodiester linkage (41 ). This linkage is resistant to most cleavage agents and therefore yields a characteristic dinucleotide upon nuclease PI digestion of spliced tRNA. However. the nucleaseresistant dinucleotide can be isolated by TLC and subsequently digested with snake venom phosphodiesterase to produce mononucleotide products. Although Protocols 9 and 10 specifically describe the analysis of splice-site junctions, these same procedures apply to the detection of modified bases. Thus methods and conditions for sample preparation, nuclease digestion, thin-layer chromatography, and quantification can be directly applied to analyses of ba~e modifications in pre-tRNA and tRNA substrates. I

1

4.3.2 Identification and quantification of splice sites Proiocol 9 describes a method for identifying the splice-site junction based on sequential digestion of spliced tRNA with nuclease PI followed by separation of the digestion products by TLC on PEl-cellulose. The nuclease-resistant dinucleotide characteristic of the splice junction is then eluted and subjected to digestion with snake venom phosphodiesterase to identify its component mononucleotides, again after TLC on PEl-cellulose. Important features of this procedure include:

(a) The use of PEl-cellulose as an effective and general means for the separation of individual digestion products; (b) Washing with ethanol to remove the solvent components from the developed PEl-cellulose plates. This is essential prior to the elution of the dinucleotide and its subsequent digestion and chromatography of the products. Washing with ethanol is effective only for LiCl-based TLC solvents. (c) The use of triethylammonium bicarbonate (TEAB) for elution of the dinucleotide from the PEl-cellulose plates. This volatile solvent can be removed by drying samples under vacuum, thus facilitating subsequent digestions and analyses. Protocol 9. Identification of splice junctions using digestion with nuclease P 1 and snake venom phosphodiesterase Equipment and reagents • PEl- cellulose thin-layer plates 120 x 20 em Cel 300 PEl plates; Macherey Nagel or Brinkman Instruments 801 503). Wash the plates in methanol then develop once

ustng water and finally dry them. Using a soft pencil, mark sample origins 1.5 em from bottom edge of each plate with at least 1 em separating individual samples

Continued 201

6: Processing of transfer RNA precursors Protocol 9 . Continued. e TLC tank (e.g. VWR ScientifiC, KT416180-0000I

• Tygon tub1ng • 32P-Iabel1ed

spliced tRNA (labelled as •n Protocols 3 or 4 : spliced and punf1ed as 1n Protocol 81

e uv light source (254 nm, e.g . F1sher Scient•f1c 11-984-201 • Cellulose powder (S1gma. C66631 • Glass m1crocapillary tubes !e.g . 1-51JI disposable p•pettes 8eckton D1ck~nson, 46141 • Sealing compound !Seal-Ease, 8eckton D1ck~nson, 1 015) • Capillary tubing ( 1 !71. Annu. Rer Brochem .. 56, :~63. 8. '\1shm1Ura. S. ( 1979). In Transfer R,\'A: wructllre-. properties and recognrtinn (ed. D Soli J \belson, and P.R. Schimmel). p. 547. Cold Spring Harbor Laboratory Preh. Cold Spring Harbor, :-.lY. 9 R1ch. ,\. and RajBhandary, U. L. ( 1976). Annu. Re1. Brochem 45. 805. 10. Kim. S -H (1979). In Transfer R.\'A: Hructure, proper/le.~ and rt•cognrtion (ed. D. Soli, J Abelson, and P. R. Schimmel), p. 83. Cold Spring Harbor Laborator) Press. Cold Spring Harbor. NY II. 'lormanl>. J. and Abel~on, J. ( 1989). Annu. Re1•. Biochem . 58. 1029. 12 Deutscher. \ 1. P. (1984) CRC Crrt Re1·. Biochem .. 17. 45. 13. Deuttol, B. L. and Greer, C. L. (1991). Nucleic ..4cids Res., 19, 1853. 4i. Kuchino, 'r , Hanyu, N., and ~t,himura, S. (1987). In .'>fethods 111 en:;;ymolog_l, Vol 155 (ed R \\ u), p. 3'"~. Academic Pre~>. San Diego. 48 Buck, M .• Connick, M .. and Ames, B. N. (1983). Anal. Btochem.. 129, I. 49. Gehrke, C. W. and Kuo, K. C., and Zumwalt, R . W. (1983). In The modified nucleoside of transfer R,\A, II (ed. P. F. Agrh, A r\. Kopper, and G. Koppe1), pp. 59, Allan R. Ll\~ Inc., '~Y . 50. Edmond>, C. G., Vestal. ~1 .. and ~lcCio~key, J. A. (1985). Suc/eic ..4ctds Res., 13, 8197. 51. Pomeranz. S. C . and McCloskey, J. A. (1990). In Methods m en~ymology, Vol. 193 (ed. J A. \lcCioskey), p. 796. Academic Pre,\, San Diego. 52. Randerath. K.• Gupta. R. C., and Randeralh. E. (1980) In \.fethods m en:..1molog;, Vol. 65 (c:J L. Gro~sman and K \loldave), p. 638. Academic Pre~s. Ne'~ l o 53. Silberklang, M., Gillum, A. t\1., and RajBhandary, U. L. (1979). In Methods ttl en;._vmology, Vol. 59 (etl. K. Moldave and L Gro~sman), p. 58. Academac Pre\\, Ne'\ Yor~ 54. Chen. J.-Y and Martin,"tic properties of group lmtrons, group ll introns, hammerhead ribozymes and hairpin ribozymes.

2. Group I intron ribozymes 2.1 The self-splitcing activity of group I introns The term ribozyme w;as coined to describe the catalytic properties of the imron in the large subunit ribosomal RNA of Tetrahymena thermophita (3). It was soon noticed that this self-splicing intron also satisfied the sequence and structural criteria for a set of introns in yeast mitochondria, designated group I by Michel et at. (4). Well over 100 group I introns have now been described (see ref. 5 for a recent compilation), most of which are in mitochondrial and chloroplast genes. However, in addition to their presence in nuclear rRNA genes of eukaryotic protists, introns of this group have also been found in genes of bacteriophage (5) and bacteria (6). Group I introns occur in genes of e'er) kind, i.e. encoding r RNA, tRNA and mRNA. Although many of these introns (like their Tetrahymena exemplar) self-splice efficiently in \•itro, others require protein co-factors (7). Howe,er, since they all share a set of structural features necessary for self-splicing (5), it is likely that all group I introns also share a common RNA-based catalytic mechanism. protein co-factors merely acting to help the RNA achieve the correct conformation. In common with the splicing reactions of group II introns in organelles (see Section 3) and snRNP-assisted pre-mRNA splicing in the nucleus (Volume I, Chapter 3), group I cleavage-ligation is accomplished through a series of trans-esterifications (Figure 1 ). Unlike the other splicing pathwars. the

7: Ribozymes ,....-..

I J El

t'~

E2

~(L~H r

~ ,r--£-2--.

EI

[

£1

£2

-.,-

..Qo,, •G - CH

+9 l;

OH

~

Q

GOH

Figure 1. Spllcong pathway and related reactoons of group I ontrons. The splicing pathw.1~ is inotoated by labelled GTP (•GTPI bond1ng on a self-splicing RNA The labelled GTP attacks the 5 ' splice JUnctoon and attaches to the 5 '-end of the ontron ( 'G - thon hnel in the forst st• ·r of splicong The 3 '-0H on the !•berated 5' exon !open box-E1 l attacks the 3 ' splice JUncttnn to join to the 3' exon (open box-E2l and form the spliced exons (open box-E 1- open box-E2 The linear lntron is released With the labelled GTP at the 5 '-end and the conserved G at th 3' end (•G-thin line-Go..) Circularization by attack of the 3 ' GOH at an internal s1te releases the labeiiEld GTP as part of a small oligonucleotode ( 'G -short thon line-0 .,). After prolongr incubat•on or at somewhat elevated pH the corcle IS attacked by hydrox1de (OH I to y od a shorter linear inrron fragment (thin llne-G OH ). Likewose for the transcnpt, at high pH or n the absence of GTP, the 3 ' splice junction is subject to attack by hydroxide ion (OH l " non-productiVe side react1on that yields the 5' exon attached to the intron (E1-thin line - 0 , I and the ~~ · exon (open box-E21

attacking nucleophile in the first step of the group I mechanism is the 3 ' -hydro:..) I group of guanosine or GTP. Consequently, the excised linear intron has an extra, uncoded G residue at its 5 ' -end. This reaction can be used to identify and characterize the self-splicing property of group I introns. Some excised introns can cyclize; the 3 '-hydroxyl group artacks a phosphc· diester bond near the 5 '-end of the intron, with elimination of a 5' oligonucleoude (3). The circular intron can then reopen, leading to additional rounds ol circularization and reduction in length. This can result m a mixture of linear and circular intron products, of various sizes, on prolonged incubation. Finably, the phosphodiester bond between the final nucleotide of the intron and the first residue of the downstream exon is especiaUy sensitive to hydrolysis. For some introns this reaction (Figure I) can be sigmficant, 212

David A. Shub, Craig L. Peebles, and Arnold Hampel even under conditions that are optimal for splicing in vitro. The product'> of this hydrolysis do not go on to splice, and so thetr presence can complicate the analysis of in vitro splicing reactions.

2.2 Detection of self-splicing introns in cellular RNA The addition of GTP to the 5 -end of excised introns (Figure I) can sometimes be used to demonstrate the presence of self-splicing group I imrons in an RNA preparation. The advantage of this reaction is that it does not require any previous knowledge of mtron or exon sequences or the identity of the interrupted gene. In practice, detection of group I self-splicing introns involves incubating deproteinized RNA, extracted from the organism under study, with [0!-3 2P] GTP and analysing the products for the presence of radioactively-labelled RNAs by denaturing polyacrylamide gel electrophoresis_ The procedure is described in Protocol I .

Protocol 1 . Detection of group I self-splicing introns8 EqUipment and reagents e 5 x GTP-Iabelling buffer 10.25 M NH4 CI, 0.15 M MgCI7, 0.15 M Tns-HCI. pH 7 5. 5 mM spermid1nel

• Total RNA 15 mg /mil, tsolated from the organism under study as described in Volume I, Chapter 1, Protocols 9-11

• 1()-"'2PJGTP 13000Ci.mmoL 10mCi·mll • Materials and equ1pment for denatunng polyacrylamide gel electrophoreSIS and autoradiography (Volume I, Chapter 1, ProtocolS or Volume I. Chapter 4, Protocol 11), for RNA samples 100-600 nt in length, a 5% polyacrylamide gel with an acrylamide:bisacrylam1de ratiO of 30:0.8 IS suitable

• Stop solution (2 5 M ammomum acetate, 25 mM sodtum EDTA, pH 8.0, 300 ~g /~1 yeast RNA ) e 0. 2M OTT • RNasm IPromega 30 un1tsl mil

Method 1. In a m•crocentnfuge tube on ice, mix the following : • 5 x GTP-Iabelhng buffer

2 (JI

e0.2 M OTT

0.25 (JI

•RNasin

0.331JI

• [ a-32P I GTP

21JI

• RNA substrate

21JI •H 2 0 to 10 Ill final volume 2 . Incubate the mixture at 37 °C for 1 h and then stop the reactton by adding 150 Ill of stop solution chilled on ice and 320 IJI of ethanol cooled to -20 ° C. 3 . Leave the mixture for 30 min at - 70 °C.

Continued

213

7: Ribozymes

Protocol 1. Contmued 4. Recover the prec1p1tated RNA by centnfugat1on for 15 mm at 4 °C in a microcentrifuge. 5. Wash the RNA pellet by resuspending 1t in 1 ml of 70% ethanol and centnfug1ng as in step 4. 6 . Repeat step 5 and vacuum dry the washed RNA pellet. 7 . Analyse the RNA products by denaturing polyacrylamide gel electrophoresis. To do this, dissolve the RNA m BtJI of H 2 0. Immediately add 8 IJI of formamide-dyes loading solution and denature the RNA for 1 5 min at 65 °C Load the sampftes onto a denaturing 5% polyacrylamide gel, run the gel at 200 V overnight and autorad1ograph 1t as described in Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4, Protocol 7 1. Autoradiographic exposures of up to 1-2 weeks might be necessary to detect weak signals. • Based on ref. 8.

The conditions in Protocol 1 are given as a guide and are not necessarily optimal. Many self-splicing im rons require incubation at higher temperatures (50 cc or more) and a buffer ''hose ionic strength is optimal for splicing in vitro. Also, since circularization eventually removes the labelled guanylate residue from the 5 '-end of the intron (Figure 1 ), the time course of the splicing reaction should be investigated. The concentration of Mg2 - ions should also be optimized; circle formation may require a higher concentration than splicing. Since RNA is readily degraded, precautions must be taken against contaminating RNases during all steps. Volume I, Chapter I (Section 1.1) and ref. 9 list general precautions that should be take:n when working with RNA. The choice of RNA isolation method (Volume I, Chapter I, Section 3) depends on the organism of interest. Naturally, the gentlest method consistent with good yield should be used. The presence of a labelled band on the autoradiograph is a good indication of a self-splicing intron. Some of the other possible imer'pretations can be eliminated by treating pan of the sample "ith alkaline phosphata~e before electrophoresis. Since group I splicing leaves the 32p.Jabelled phosphate exposed on the 5 '-end of the excised intron, it should be removed by the phosphatase. In addition to the excised int ron, the intermediate splicing product, consisting of the intron plus the 3 '-exon (Figure 1), can sometimes be seen as a second labelled band of lower electrophoretic mobility. More than two prominent bands may indicate multiple introns (see ref. 10 for an example). Thus, a positive result can reveal the presence of a group l intron and allow its size, and that of the downstream exon, to be calculated. However. failure to detect end-labelled RNA in this assa) cannot be considered proof of the absence of group I intron.s, since the assay can fail to detect group I introns for various reasons including: 214

Da,·id A. Shub. Craig L. Peebles. and Arnold Hampel • the requirement for cellular components to facilitate '>pi icing in t•itrv ethe low abundance of unsphced precursors in the ~:ell, because the introns are located in rare tran~cnptl> • the RNA precursors '>plice so readily m vivo that the concentration of self-splicing substrates in the RNA isolated from the organism is too lo'' for detection

2.3 Confirmation of self-splicing by in vitro transcription of

cloned DNA containing putative group I introns -\na1ysis of the products of in t·uro transcnption can provide e\idence for self-splicing of cloned putati\e introns . If self-splicing occurs efficiently in ~·itro, spliced products corresponding in size to the ligated exom and to the excised intron should be detectable in addition to the prima[) run-off transcript. For this assay, the DNA fragment containing the putative group I 1ntron has to be cloned into a vector which contains promoters for RNA polymerase adjacent to the cloning site. There are many commercially-avallable plasmids which can be used that contain multiple cloning sHes flanked on either side by promoters for SP6, T3, or T7 bacteriophage RNA po)ymerases. U~e of both the 5 and 3 ' flanking promoter~ aiiO\\\ transcription of the cloned insert in either direction. Since prematurely-termmated or partiallydegraded transcripts can complicate the analysis of RNA products arising from splicing, the in vitro transcription mixture should be run on a denaturing polyacrylamide gel and the full-length transcript isolated by elution (Volume l, Chapter I, Protocol 8 or Volume l, Chapter 4, Protocol fJ ). In addition, the authors recommend that the template should not exceed 1-2 kbp for efficient transcription into a full-length product, best reslllt~ being obtained ,.,.ith the shortest transcripts possible. Therefore, run-off transcripts should not extend into the \ector sequences. This can be ensured by truncation of the DNA template at the multiple cloning site or within the cloned msert by digestion with suitable restriction enzymes. Volume r. Chapter I (Section 2.2) and Chapter 2, this volume (Section 2.6} give additional information about in vitro transcription method~ . Protocol 2 describes the in vitro transcription and analysis of sequence~ containing group I self-splicing introns. Templates based on pBS-M 13 • (Stratagene) and isolated by the alkaline lysis mini preparation method (II) without RNase treatment are usually adequate for m l'itro transcnption. The DNA samples should be linearized by restriction digestion and then extracted with phenol and ethanol precipitated. Contamination with RNases must be avoided during the in ~·itro transcription and subsequent steps; the reaction mixture described in Protocol 2 contain:. RNasin but other precautions should abo be taken as advised in Volume l, Chapter I, Section 1.1. The GTP concentration present in the transcription reaction mL\ture is sufficient to allow self-splicing of group I mtrons. 215

7: Ribozymes

Protocol 2 . In vitro transcription of cloned group I introns

-, I

Equipment and reagents

• la-32P)UTP

(20 mCo/ml, 800 Co/mmoll

• 5 x transcnption buffer (0.2 M Tns-HCI, pH 8.0, 40 mM MgCI 2 • 10 mM spermodine 0 .25 M NaCII e o . 75 M

on

• RNasin (Promega, 30 units/ml) • 0.5 mM UTP • 10 mM stock solutoons of ATP. CTP, and GTP

• RNA polymerase (50 unots J.ll) from T3 or T7 phage depending on the nature of the vector used e DNA template (0 2 - 0. 51-'Q II-'11 contaoning the cloned sequence of interest down· stream of a T3 or T7 promoter • Materials and equopment for denaturing polyacrylamide gel electrophoresos and autoradiography (see Protocol 1 )

Method 1. Make up the following reaction m1xture (sufficient for 8 transcription reactions) at room temperature to avoid forming a precipitate: • 5 x transcnption buffer e o 75M OTT e RNasin

8 JJI 81JI 1 J.ll

e 10mMATP

1.6 1JI

e lOmM CTP

1.6 J.ll

e 10 mM GTP

1.61JI

e 0.5 mM UTP e [ a-32 P] UTP

1.6 t.JI

1.61JI •H 20 13 t.JI 2. Add 0.4 JJI of T3 or T7 RNA polymerase and then div1de the reaction mixture into 4 IJI aliquots. 3. Start each 1nd1vidual reaction by adding 1 IJI of DNA template. 4. Incubate the tubes at 37 ° C for 1 h. 5. Stop the reactions by add1ng 8 JJI of formamide-dyes loading buffer to each tube. 6 . Denature the samples by heating them at 65 °C for 1 5 min before analysing them on a denatunng polyacrylamide gel as described in Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4 , Protocol 11

L---------------------------------------------------~

If imrons are vel") actively spliced during in ~·irro transcription, pn:cursor molecules may not accumulate to high levels. In such cases, the authors carry out the transcription reaction (Protocol 2) in the presence of very high concentrations of nucleoside triphosphates (typically 2 mM of each) leaving the concentration of MgCI2 unchanged. This reduces the effective Mg2 ion concentration below the level needed for splicing, while still allO\\ ing efficient transcription. 'IJaturally, some adjustments of the NTP and M·g2 + ion 216

David A. Shub, Craig L. Peebles, and Arnold Hampel

concentratiom may be required to achieve the best compromise between high rates of transcription and lO\\o levels of splicing. Since all of the molecular spec1es illustrated in Figure I rna} be among the products of a single transcription reaction, electrophoretograms can be difficult to interpret. If the size of the Linear intron is already known from the GTPlabelling assay (see Protocol I), a band of corresponding mobility should certainly be present on the autoradiograph. If a physical map of the insert is available, the insert can be truncated to different lengths using appropriate restriction enzymes. AnalySIS of the in vitro transcripts and spliced products from these modified templates can be used to deduce the position of the intron within the original insen:. No spliced products should be detected from transcripts ending \\oithin the intron. Similarly, deletions between the promoter and the intron can reveal whether any of the bands are due to premature termination of transcription. Ultimately, the nucleotide sequence of putative ligated exon species must be determined and compared to the DNA sequence in order to characterize fully the splicing event. Full characterization of the self-splicing reaction requ1res purification of the precursor transcript, generally accomplished by denaturing polyacrylamide gel electrophoresis and elution (Volume I, Chapter I, Protocol 8 or Volume I, Chapter 4, Protocol I I). Precursors may be uniformly labelled, in which case non-radioactive GTP is substituted to start the reaction (see Protocol 1). This discussion has been limited to the group I ribozyme reaction involved in R 'A processing, namely splicing. There are, however, a large number of other reactions that have been demonstrated on cleverly-engineered intron ribozymes. The interested reader should consult Cech {I 2) for a review of several of them.

3 . Group II intron ribozymes 3.1 Identification of group II introns Group II introns were first recognized as a specific class of inten ening sequences m yeast mitochondrial genes that are disunct from the group I introns (ref. 4 and reviewed in refs 12 and 13) Each group of mitochondrial introns is different from nuclear pre-mRNA introns, based on a comparison of the boundary sequences and internal structures. Group I or group II introns may be distinguished by their characteristic secondary structures and conserved sequences (13, 14) Standardized secondary structure diagrams for group I and group II introns have been reviewed recently (13, 15). These generalized structures can accommodate the many newly-identified members of each group. Furthermore, each group can be divided into subgroups according to specific structural features that are not general for the entire group. Thus these introns represent a diverse and expanding family of molecules. A detailed description of group II intron structure is gi\en in Section 3.2. 217

7: Ribozymes

S2.;

A. TRANS-ESTER l F l CAT I ON

B. HYDROLYSIS ~ 'A

'A~cri pt (GCGAG < • ) r2Z:1

1

1

H ·o·1

1

STEP 1

k ansesterification

1

~

, linear 2/3's GCGAGP W-)e r mediate 't I

s

~

STEP 2

transeosh•rification

(GCGA I~+ V/21 1, lariat i ntron spliced exons

STEP 1

hydrolysis

~

exonl

exonl ~ ~ 2/3's lariat G...-L-"-)e r mediate (GCGA I

S G.rA

transcript

(GCGAG ~

GCGAGP

t

1

STEP 2

transesterification

,+V/21

linear i ntron

I,

spliced exons

Figure 2. Self-splic1ng pathways of group II introns . The sequence used throughout is that of the group II self-splicing intron a 5; of yeast mitochondria lA) The standard self-splicing pathway is in1t1ated by rrans-estenflcation The first step is attack by the branchpoint adenosme (AI on the 5' splice junct1on to release the 5' exon (hatched box) from the 2/3's lanat intermediate composed of the 1ntron (GAGCG-thin line) and the 3' exon (open box). The second step IS attack by the 3'·0H of the 5 ' exon on the 3' splice Junction to release the spliced exons and a lanat intron. (8) An alternative self-splicing pathway is initiated by hydrolysis and is thus uncoupled from branch1ng. The first step is attack by water 1"0") on the 5' splice junction to release the 5' exon (hatched box) from the linear 213's intermediate composed of the mtron (pGAGCG-thin lme) and the 3' exon (open box). The second step is attack by the 3'-0H of the 5 ' exon on the 3' spl1ce junction to release the spliced exons and a linear mtron. The arrowheads 1nd1cate the 3' direction or a 3'-0H end

Group II introns have been identified in the mitochondrial or chloroplast genomes of fungi, plants and protists (13). Like many group 1 introns (reviewed in ref. 12), some group II introns are capable of self-splicing in vitro (16-21). For group II introns (Figure 2A ), the first step is an attack on the phosphate group at the 5 ' splice junction by the 2' -hydroxyl group of an unpatred adenosine near the 3' splice junction. This trans-esterification reaction releases the 5' exon with a free 3 ' -hydroxyl group and creates a 'lariat' intermedtate In the second step, the 3 '-hydroxyl group of exon I attacks the phosphate group at the 3 ' splice junction to create the spliced exons and release an excised intr<m lariat. By analogy with group 1 intron ribozymes (Section 2) and other transesterification reactions, group ll ribozymes are presumed to position a Mg~ + ion as an activating electrophile and to fold specifically for alignment of the reactants to promote an in-line attack on the phosphorus atom. While the primary products of the group 1I self-splicing reaction are linear spliced exons and excised lariat introns (see Section 3.4 and Figure 2), several other 218

David A. Shub, Craig L. Peebles. and Arnold Hampel

IBS21 Exon 1

Exon2

Figure 3. Schematic diagram of group II intron secondary structure. A stylized drawing after the model of ref. 13 showing the secondary structure of a group II intron as the thin line; the curved segments are identified as unpa1red 1n the secondary structure with parallel stra1ght lines indicating helical segments. The 5' exon and 3 ' exon are indicated as heavier straight lines. Sites involved in tertiary base pairing interactions are labelled and mdicated with the grey highlight bars (EBS 1, EBS2, IBS 1, 1852, a , a ', 13. 13', E.£' ). Each domain and subdomain (within domain 1) is indicated, and the other features are discussed in the text (Section 3.2) No attempt has been made to diagram the secondary structures of domains 2 and 4.

splicing-related reactions have been described (see Section 3.5). This section will focus on the various conditions that have been used to analyse splicing and other reactions of group II introns.

3.2 Group ll intron secondary structure and functional

anatomy The distinctive secondary structure of group 11 introns is usually displayed as a 'cloverleaf' consisting of six 'domains' numbered 1-6 (Figure 3 ). Each domain is bounded by 'pairing segments' that are represented as helical stems. Each domain is separated from its neighbours by shon ' joining segments'; joining segments are conserved in length and partly conserved in sequence (13). The 5 1 splice junction is apparently selected by tertiary base-pairing interactions between exon and intron sequences. There are two exon-binding sequences (EBS I and EBS2) in subdomain D of domain I; EBS I and EBS2 are complementary to intron-binding sequences (JBS I and IBS2) in the 5 1 exon (exon I). These pairings are designated IBSl :EBSJ and IBS2:EBS2 (22). 219

7: Ribozymes

While the exon sequences adjacent to the splice sites are highly ,·ariable, there are recognizable consensus sequences defined for both 5' and 3 ' termini of the intron. The 5 ' consensus is-! GUGCG- and the 3 ' consensus is -A Y l-, where l designates the splice site (13). A base-pairing contact designated E:E' involves nucleotides near the 5 ' 1ntron boundary pairing with an internal loop of subdomain C I of domain 1 (23). Based on phylogenetic co-variation, two additional long-range base-pairing contacts are predicted to occur ,.,ithin domain I, designated cx:cx and ~:W (22, 24). Because of its complex internal structure and demonstrated role in the spectl t~ recognition of exon 1 (22), domain 1 is generally believed to contribute much to the catalytic core of group II introns. Domain 2 is rather variable in size and sequence and has no known specific role in group II activity. Domain 3 is more uniform in size, but, like domain 2, has not yet been assigned an · specific function. Domain 4 is extremely 'ariable in size and sequence, contains a large open reading frame in some group II imrons, and may be entirely deletea without severe consequences for self-splicing activity (25). Domain 5 is the mos highly conserved in both size and sequence of any domain; the presence o domain 5 may be considered diagnostic for group II imrons (13). Domain " is critical for function in vitro, playing an essential role in the first transesterification step of splicing (25), although the details of domain 5 interaction with other parts of the intron are not yet characterized . Domain 6 contains the unpaired adenosine that is the site of branch formation ( 17) and consequently may be considered one of the substrates of the ribozyme. Deletion or alteration of domain 6 can prevent branch formation, but splicing still proceeds in vitro (26-28) .. So far, there are no extensive base-pairing interactions defined between the intron and the 3 1 ex on (exon 2). However, the nucleotide on the 5 1 side and adjacent to EBSl pairs with the first nucleotide of exon 2 (28). This interaction has been designated the ' internal guide' (ig:ig' ). The last base of the intron participates in a solitary basepair with a base in J3 ; this contact is termed ,.:;' and affects the rate of the second trans-esterification step of splicing (23). In addition to ,:,· , domam 6 is needed to promote accurate and efficient reaction at the 3 1 splice junction (28).

3.3 Pl[)tential of group IT intron ribozymes for practical use Group ll introns are capable of a variety of potentially useful reactiontrate to initiate the reactio n Reactions of group I introns have the distinct advantage that their cleavage products can be labelled specifically by the addition of GTP (see Protocol I) Nevertheless, group II introns offer high specificity, and research is continuing for improvement of group II introns as nbozyme reagents for RNA cleavage 220

David A. Shub. Craig L. Peebles. and Arnold Hampel

and recombination. At present, there are no published reports for applications of group II introns as ribozyme agents for down regulation of gene expression

3.4 Self-splicing of group II introns 3.4.1 Introduction Some group ll introns are capable of self-splicing m vuro ( 16-21 ). The earliest examples were a5r- and bl, both of which are derived from the mitochondrial genome of the yeast Saccharomyces cerevisiae (16-18) and are members of subgroup llB (13). More recently, examples of subgroup IIA imrons, including al and a2 from yeast, have been shown to be self-splicing under some conditions in virro ( 19-21 and P. S. Perlman, personal communication). Most studies of mutants and alternative reactions have focused on as.., and bl. AU group II introns are candidates for self-splicing, but it should be pointed out that selfsplicing has not been demonstrated for every group 11 intron tested. Several protocols are currently in use by various laboratories studying group 11 introns. The original reaction conditions were devised for the prototype of self-splicing group II introns, a51' of yeast (16). The splicing conditions were similar to those for in vitro transcription of the RNA substrate, since spliced products were detected among the transcription products. Subsequent!>, modifications were made to improve the rate and yield of the ribozyme reactions. The following sections (Sections 3.4.2-3.4.4) describe the synthesis of ribozyme substrates, tht! basic procedure for studying self-splicing of group I I imrons, and variations on this basic method. Later sections (3.5.1-3.5.3) discuss other reactions of group II imrons. 3.4.2 Preparation of transcripts for self-splicing In order to detect self-splicing of a knO\.,n or suspected group II intron, it is advantageous to prepare reasonable amounts of homogeneous transcripts spanning the intron and including both exon I and exon 2 segments. This may be conveniently accomplished by cloning a restriction fragment or an appropriate PCR-amplified DNA segment containing the imron into the multiple cloning site of a plasmid with a promoter derived from SP6, T3, or T7 bacteriophage. At least 50 nt of the natural exon 1 sequence should be included to ensure that the entire length of IBS 1 and IBS2 are included. No more than a fe,, bases of exon 2 are needed. Additional sequences derived from the plasmid can be retained at either end, usually without posing problems m the subsequent transcription or self-splicing reaction. It is frequently convenient to have the total length of the exons similar in size to the intron; then the spliced exons, separate exons, and intron products are easily displayed on a single gel. To prepare the template for in vitro transcription, purified plasmid DNA must be cut at a restriction site downstream from the inserted fragment to define the 3' -end of the transcript. Restriction enzymes that leave blunt ends or 5 e:-.temions are preferred to those leaving 3' extens1ons. Cleavage withm 221

7: Ribozymes

the mtron will prevent splicing, but some intron fragments retam ribozyme activit}'. Care must be taken to eliminate traces of RNase typically used n plasmid preparations. An alternative method of preparing transcriptto 1 templates is to use PCR to amplify the exon-intron s.equence with an appropria:e promoter sequence built into the upstream primer. In vitro transcripuon is carried out as described in Volume I, Chapter I, Protocol 4. A procedure is also given in Protocol 2 of this chapter. Becau!>e yeast group II introns contain 40% uridylate residues, it is most convenient to label transcripts using [ a-32P) UTP . However, any suitably labelled nucleottde containing 3H, 14C a-35 S, or a -32 P should be successful. Since group II intron transcripts are typically over I kb in length, transcription conditions must be optimized with respect to the yield of homogeneous full-length products. Good quality RNA polymerase (United States Biochemicals) and ribonucleoside triphosphates (Pharmacia) seem to be important in this respe~t. Otherwise, full-length transcripts must be purified b:y gel electrophore~is . This may be done using 40Jo polyacrylamide gels with 0.1 OJo SDS and ~.\I urea run with TBE buffer as described in Volume I, Chapter I, Protoco/8 or Volume I, Chapter 4, Protocol 11. After the product bands ha\e been identified by autoradiography or UV shadowing, the RNA can be eluted from the gel by soaking overnight in 0.5 M ammonium acetate, 5 mM EDTA. However, bener yields and more rapid recovery are obtamed by electroeluuon in either Tris-acetate-EDTA or TBE buffers with a Schleicher and Schuell Elutrap device. After transcription, either with or without gel purification, the RNA is recovered by ethanol precipitation and dissolved in water at a concentration of I03 -104 c.p.m./IJI. The transcripts should be stored

at -20 oc.

3.4.3 The self-splicing reaction The basic procedure for self-splicing of group II introns is described in Protocol 3. It is convenient to set up the splicing reaction by adding one volume of labelled transcript to an equal volume of 2 x sphcmg reaction buffer. For intramolecular splicing assays, the concentration of the transcript is not crucial; the amount of RNA used is determined by its specific radioacti\'it) and the amount of radioactivity needed to produce an acceptable autoradiographic exposure time. The aULhors routine!} use J()C 105 c.p.m. of the transcript in a I0 IJI reaction volume. Larger or smaller reaction volumes rna} be used a~ dictated by the particular circumstances. The incubation temperature is important, since· the temperature profile is rather abrupt for self-splicing by all known group II introns (for example, ~ee ref. 16). The optimum temperature in vitro is typically 40-50 °C, with the best temperature depending on the intron. Incubation times are typically 0.5-4 h at 45 oc. When investigating the self-splicing of a group II intron for the first time, the optimal incubation temperature and reaction time must be determined in pilot experiments. 222

David A. Shub, Craig L. Peebles, and Arnold Hampel The products of the self-splicing reactton are analy~ed b> electrophoresi~ on a 40!o polyacrylamide gel "ith 0.1 cro SDS and 8 "\1 urea run in I x TBE buffer. Under these conditions, linear R~As of 0.1-2.0 kb can be resolved reasonably well. Typical runnmg times for a 30 em gel are 2-4 h at 700 V. For the large)t group II introns, 3'ro or 3.51ro polyacf)lamide gels may be more appropriate. Non-hnear RNAs, such as the lariat excised intron. display great!} retarded mobility compared to the linear form of the same total length. Consequently, the product most characteri~tic and clearly visible after ~uccessful self-splicing is the lariat intron product, since it often mtgrates more slowly thalli the starting transcript. However, the relattve mobility of the lariat varies somewhat with the degree of cross-linking of the gel matrix, the buffer salt concentration. the denaturant concentration, and the gel running temperature. Protocol 3. Basic self-splicing reaction for group II introns Equipment and reagents • :..t, although thh. has yet to be demonstrated conclusively. The specificit> of these hydroly~;is reactions is defined largely by EBS I, a sequence block of about si\. bases. Amazing!~. imron RNA~ will also catalyse hydrolysis of the ~pliced exon product (34). This reaction has been designated 'spliced exon reopening' or 'SER' (see Figure 4£) to distinguish it from the similar SJH reaction. There are sevl"ral indications that SER and SJH are different events. For example, the group IIA imron a I of yeast carries out both SJH and self-splicing, but is inactive for SER (21). Similarly, deletion of domain 3 from a5"1 results in substantial reduction of the SER reaction while self-splicing and SJ H are much less affected (31 ). In applications of the group II ribozyme, the SJH activity acting in trans rna} be the most useful, since maximal cleavage specificity is highly predictable based on the sequence of EBSI . The role of EBS2 is not well unde·rstood, but it evidently contributes to effictent hydrolysis in trans (36). The IBSI and IBS2 target sites may be dtrectly adjacent or separated by a fe,, nucleoudes. However, only a few different sequences are available at present on which to base these predictions.

3.5.2 Splice junction hydrolysis and spliced exon reopening reactions RNA substrates for SJH and SER may be synthesized by m vitro transcription of an appropriate DNA template in the presence of [cx-lZP]UTP (see Protocol2 and Section 3.4.2). For example, exon I with domains 1-5 (El: 12345) "ill undergo SJ H efficiently (33). lntron lariat and spliced exon products are needed for SER (34). For the ribozyme reaction, mix the transcript with an appropriate high-salt reaction mixture, such as high KCl (see Table 1). For SJ H with tlhe E I: 12345 transcript, the RNA concentration is not important. Incubate the reaction for 225

7: Ribozymes

A . SJH (cis)

C. SJH* ( example 2)

~ transcri pt 'l!tith OS

1

(GCGAG ~ H-o-'~1

hydr olysis

1

H-o·~l

~+ ~CGAGP ~ exon 1

1

i ntron fmgment

B. SJH*

QG 'A~

~ GCGtl '--+-~ 1 1ntron - exon 2 t xt H-o·~l

__ll ' AS.

+

~

1

H.0.H

c+

G 'A=> GCGA P ~

't

1 -J

i ntron

+

~

GCGAG ~ (

fragment

hydrc1lys is exon 1

~

exon 2 f r agment

~~ 3 ' tntron

exon 1 pG.. tx t hydrolysis

+

.As

0. SJH (t rans)

1 ft

+

GCGAGP ~ 5' i ntron fragment

f--,

( exampl e 1)

hydrolysis

1

pG-+ fregment

E. SER ( trans)

I

V//1

tnt ron -exon 2 fragment

I I+ H.o.H

A s

spliced exons hydrolysis

j

(

GCGAG.r~ 1

lari at i nt r on

~+CJ...

exon 1

exon 2

Figure 4. Sphcmg-related ribo1•yme react1ons of group II mtrons. The exon and intron segments are represented by the same conven tions as used m Figure 2: the hatched box represents exon 1, the GAGCG-thm line 1s the mtron, the open box is exon 2. and the arrowheads 11re the 3'-0H end or the 3' direc:t10n. (AI Splice junction hydrolysis (SJH (cis!] is outl1ned tor a transcript extending through domain 5. (6) Splice junction hydrolysis occurs With a relaxed spec1fic1ty [SJH • (example 1)] at a non-canonical site (arrow) in the 3' exon using a transc11pt extendmg through the intron and 3' exon (intron-exon 2 txt). (Cl Splice junction hydrolysiS also occurs With a relaxed specificity [SJH • (example 21 J at a non·canonical site (arrowl 1n the intron usmg purified linear intron. (0) Splice JUnction hydrolysis [SJH (rrans)] is shown for an intron transcnpt extendmg through domam 5 that acts on a second transcript conta1n1ng the 5' splice junction (exon 1 txt). (EJ Spliced exon reopening [SER (trans)) is shown for an intron lanat actmg on thll purified spliced exons.

226

Da,•id A. Shub. Craig L. Peebles, and Arnold Hampel

A . TRANS- SPLICING

B. TRANS-SPLICING

( example 1) ~ exo n 1 pG-+ txt

+

( example 2)

+'A~

H.0 .H

__..0_.

:.:?

STEP 1 QG 'AS . hydro- (GCG~ ~ lysts ....-:---+•-----:,-' 1ntron - exon 2 txt

1

1

__..0_.

exon 1

~ -AS

(-GCG~ fi~ ,_J

+

1ntron - exon 2 txt

STEP 1

tr ansesterification

e~~A~

pG-+ fragment

(GCGAG-"~

Y-intron intermed1ate

1

STEP 2

1

tr ansestt'nficatlon

V/ZJ +

r?Lzr==J,

I,

sph ced exons

STEP 2

transesterification

Q 'A~

( GCGAG

s pl'i ced exo ns

GCGAG-"~

+

~

As

(

linear' i ntron

- 1·

_;

Y- intron

Figure 5. Trans-splicing react1ons of group II 1nrons. The exon and intron segments are represented by the same conventions as used in Figure 2: the hatched box represents the 5' exon (exon 1). the GAGCG-th1n lme is the intron, the open box IS the 3' exon (axon 2). and the arrowheads are the 3 '-0H end or the 3 direct•on. (A) One example of rrans-splicmg requ~res an axon 1 transcript and an 1ntron-exon 2 transcript. (B) A second example of trans-splictng requires one transcript extending through doma•n 3 (5' txt) and another transcnpt containing domams 5 and 6 and exon 2 :3 txt)

10-30 min . For efficient SER, each transcript should be used at a concentration of about 0. 1 ~ M and incubated for 30-120 min in the high KCl buffer (see Table 1 ). After the ribozyme reaction, purify the products by electrophoresis and autoradiography as described in Protocol 3.

3.5.3 Trans-splicing reactions of group

n

introns

Some unusual group n introns ha\.e been identified that are split mto two or more transcripts (37 -40). These separate RNAs presumably associate to form the correct ly-folded acti"e intron and undergo splicing in trans (see Figure 5A, B). In vitro models of such trans-splicing reactions have been constructed and used to analyse the requirements fo r efficient binding and catalysis. The first such model sy,tem supplied exon I as a transcript with a shon J extension to a second 227

7: Riboz~·mes transcript that contained the entire intron and exon 2 sequences (36). These tramcripts associated through lBSI :EBSI and IBS2:EBS2 to allow SJH and step 2 of splicing. No branching was detected. The major products \vere spliced exons, a short linear mtron fragment and a full-length linear intron. Producm : reactions occur under several buffer conditions, but with a requirement for high RNA concentrations such as 0.1 JJM for each participating molecule. This is reasonable for a second order system limited by RNA: RNA association. Later, trans-splicing systems were established between transcripts that each contained some of the intron domains and one exon (25). In effect, the self-splic 1g transcript was divided v. ithin a domain or between two domams. As befor· the most efficient reactions were observed at high concentrations of each Rl\ \ in the range of 0.1-1.0 JJ~l for each half-molecule. This style of trans-splicir .7 occurs best in the high KCI or high ammonium sulphate reaction buffers (~ee Table I) . The products are spliced exons and a Y-shaped excised intron; some separate exons and linear intron halves are also produced as byproducts. The'e trans-splicing systems have been useful for examining the roles of individual domains in the overall reactivity of the intron. For example. this approach \\ as used to show that domain 4 is entirely dispensable, while domain 5 IS essemial for splicing (25). The trans-splicing reaction is carried out using a procedure similar to that for self-splicing (see Protocol 3). First, prepare transcripts for the two halve~ of the intron, for example exon 1 with domains 1-3 (E I: 123) and domains 5-6 w1th exon 2 (56:E2). Use in ~·irro transcription of appropriate DNA templates (SecLon 3.4.2). Purification of the transcripts by electrophoresis may not be necessary. The rate and extent of the trans-splicing reaction depend on the concentrations of both reactants. Each should be present at a high concentration (at least 0.1 JJM). Small reaction volumes are useful to achieve these high concentrations: use a 4 JJl reaction volume overlaid with mineral oil to prevent evaporation. L ~e an appropriate high-salt buffer (see Table 1) and incubate the reaction mLxture for 30-120 min at 45 °C. Analyse the yield of products by electrophoresis and autoradiograph} (see Protocol 3).

3.5.4 Reverse splicing and transposition reactions The total number of phosphodiester bonds is comerved during the group II self-splicmg reaction. Therefore, each step and the entire reaction should be readily reversible. Recently, reverse reactions, where the intron lariat insert~ at the product splice junction, have been described and analysed in some detail (41, 42). Figure 6 illustrates examples of reverse splicing. imprecise 'rever~nr events have also been discovered, where the intron lariat inserts at sites related tO IBSI found within an otben\ise unrelated transcript (31, 42). Such insertion products can be 'preserved' b) reverse transcription and eDNA clonin.5. Transcripts of the resulting clones are then capable of fonvard splicinE (31 ). The reverse reactions have been termed RNA recombination or RN \ transposition (31, 42). 228

D01·id A Shub. Craig L. Peebles. and Arnold Hampel A. REVERSE SPLICING

c.rA~

(GCGA

I ~+V/21

leriet 1ntron

1

B. BRANCH ADDITION

cA~~

(GCGA 1 ~+~

I

spliced exons

1eriet i ntron

REVERSE

1

STEP 2

exon 1

REVERSE STEP 1

trans.sttnf"1Cation

transu"t•rifieation

___D_.

exon 1

~ ~ 21.3·; hriet

GCGAG.r~ermediete

(

I

1

REVERSE STEP 1

tr anststtrif1eation

C. PARTIAL REVERSAL

W 'A~ngth GCGAG ~ecursor (

GCGAGP

\

I

'A~ ) Lid., Bell Lane. Lewes, East Sussex BN7 lLG. UK. Boehringer Maonheim Corporation, BiochemtcaJ Products, PO Box 5041-t, lndianapohs, IN -t6250-0413. uSA Braun (B. Braun l\1elsungen AG) , PO Box 120, D-3508 \.telsungen, German) Braun (B. Braun Medical Ltd) , 13- 14 Farmborough Close, Aylesbur} \'aJc Industrial Park, Buck~ HP20 JDQ, UK. Brinkman Instrument Co., Cantigue Road, Westbury, "'Y 11590, USA. c/ o Chemlab Instruments Ltd., Hornminster House, 129 Upmin~ter Road, Hornchurch, Essex, UK. Calbiochem, PO Box 12087, San Diego, CA 92112-4180, USA. Calbiochem-Novabiochem (UK) Ltd., 3 Heathcoat Building, Highfields Science Park. University Boule\ard, Nottingham NG7 2QJ, UK. Cornjng Medical and Scientific Glass Co .. Medfield, \1A 02052, USA. Ciba Corning Diagnostics, Colchester Road, Halstead, Essex C09 2DX. UK. Difco Laboratories Ltd., Central Avenue, East Mole ey, Surrey KT8 OSE, Ll . Difco Laboratories, PO Box 331058, Detroit, Michigan 48232-7058, USA Du Pont Co. (Biotechnolog) Systems Di\·ision), PO Box 80024, Wilmington, DE 19880-0024, USA. Ou Pont UK Ltd., Wedgwood Way, Stevenage, Hens SGI 4QN, UK. Eastman Kodak Co. , PO Box 92822, LRPD-1001 Lee Road, Rochester, NY 14692-7073, USA. c/ o Pha~e Separations Sales, Deeside Industrial Park, Deestde, Clw)·d CH5 2NU, UK. Falcon; contact Becton Dickinson. Fisher cientific Co .. 50 Fadem Road, Springfield, NJ 07081, USA. Flo\\ (ICN Flow), Eagle House, Peregrine Business Park, Gomm Road, High Wycombe HP13 7DL, UK. Flo\\ (lC:-.. Biomedjcallnc.), 3300 Highland Avenue, Costa \lesa. CA 92626, USA. Fotodyne Inc. , 16700 West Victor Road, New Berlin, Wisconsin. USA. Gibco BRL (Life Technologies Ltd), Trident H ouse, Renfrew Road, PaiskY PA3 4EF, UK. Gibco BRL (Life Technologies Inc.), 3175 Staler Road, Grand Island, :-. Y 14072-0068, USA. Gilson France SA, BP 45, 72 rue Gambeua, 95400 Vilbers le Bel France. c o Anderman L!d .. 20 Charles Street, Luton, Beds LU2 OEB. L"K. Hoefer Scientific Instruments, PO Box 77387-0387, 654 Minnesota Street. San Francisco, CA 94107, USA. Hoefer UK Ltd., Newcastle, Staffs ST5 OTW, UK. 242

Appendix 1 H~Cione Laboratories, 1725 State Highway 89-91, Logan, UT 84321. USA. IBI (International Biotechnologies Inc.), PO Box 9558, e\\ Haven CT 06535, USA . IBI Ltd., 36 Clifton Road, Cambridge CBI 4ZR, UK. ICN Biomedicals; see Flow (ICN). ISCO Inc., PO Box 5347, Lincoln, NE 68505, USA. c/ o Jones Chromatography Ltd., Tir-y-Berth Industrial Estate, Ne\\ Road, Hengoed, !\tid Glamorgan CF8 8AU, UK. lsolab Inc., PO Box 4350, Akron, OH 44398-6003, USA. c/ o Genetic Research Instruments Ltd., Gene House, Dunmo\\ Road, Felstead, CM6 3LD, UK. Kimble Products, 1022 Spruce Street, Vineland, NJ 08360, USA. Kodak; see Eastman Kodak. Kontes Glass Co., Vineland, NJ 08360, USA. c/o Burkard Scientific Sales, PO Box 55, Uxbridge, Middx UB8 2RT, UK. M.A. Bioproducts (Microbiological Associates), Biggs Ford Road, Building 100, Walkersville, MD 21793, USA. c/ o Lab lmpex Ltd., Waldergrove Road, Teddington, Middx., T\Vll 8LL, UK. Macberey Nagel, Postfach 307, Neumann-Neanderstrasse, D-5160 Duren, Germany. c/ o Camlab Ltd., Nuffield Road, Cambridge CB4 ITN, UK. Millipore lntertecb, PO Box 255, Bedford, f..IA 01730, USA. MiJJipore UK Ltd., The Boulevard, Blact...moor Lane, Watford, Hens WDI 8YW, UK . Nalge Co., PO Box 20365, Rochester, NY 14602-0365, USA. c/o FSA Laboratory Supplies, Bishop Meadow Road, Loughborough, Leics. LEI I ORG, UK. New England Biolabs (NBL), 32 Tozer Road, Beverley, fvlA 01915-5510, USA. New England Nuclear (NE~), Du Pont Co., NEN Research Products, 549 Albany Street, Boston, r-.1A 02118, USA. DuPont UK Ltd., Wedgwood Way, Stevenage, Herts SGl 4QN, UK. Pharmacia Biosystems Ltd. (Biotechnology Division), Davy Avenue, Knowlhill, Milton Keynes, MK5 8PH, UK. Pharmacia-LKB Biotechnology Inc. , PO Box 1327, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA. Pierce, PO Box 117,3747 North Meridan Road, Rockford, 1L 61105, USA. c/o Life Science Labs Ltd., Sedgewick Road, Luton, Beds LU4 9DT, UK. Pierce Europe BV, PO Box 1512,3260 BA Oud-Beijer1and, The Netherlands. Promega Ltd., Delta House, Enterprise Road, Chilworth Research Centre. Southampton SOl 7NS, UK. Promega, 2800 Woods Hollo'' Road, ~ladison, WI 53711-5399. USA. Qaigeo Inc., Studio City, CA 91604, USA. c/ o Hybaid Ltd., 111-113 Waldegrave Road, Teddington, Middx T\\ II 8LL. UK.

243

Appendix 1 Sartorius AG, Postfach 32-43, Weender Landstrassc 94-108, D-3400 Gottmgen, Germany. artorius Ltd., Longmead, Blenheim Road, Epsom, Surrey KT9 9Q , UK. Sartorius ~orlh America Inc. , 140 Wilbur Place, Bohemia, Long Island, N"\ 11716, USA. Schleicher & Schuell, Postfach 4, D-3354 Dassell, Germany. c/ o Anderman & Co. Ltd. , 145 London Road, Kingston-upon-Thames, Surrey KT2 6NH, UK. Searle; contact Amersham. Sigma Chemical Co. Ltd., Fancy Road, Poole, Dorset, BH17 7NH, UK. Sigma Inc., PO Box 14508, St Louis, MO 63178, USA. Sorvall; contact Du Pont. Squibb Pharmaceuticals , I Squibb Drive, Cranberry, NJ 08Sl2-9S79, USA. Stratagene Inc., 11011 North Torrey Pines Road, La Jolla, CA 92037, USA. Stratagene Ltd., Unit 140, Cambridge Innovation Centre, Milton Road, Cambridge CB4 4FG, UK. United tales Biochemical (USB) Corporation, Box 22400, Cleveland, OH 44122, USA. c/ o Cambridge Bioscience Ltd., 25 Signet Court, Stourbridge Common Business Centre, Swans Road, Cambridge CBS 8LA, UK. Unhersil)' of Wisconsin, Genetics Computer Group, University Avenue, Madison, WI 53706, USA. UV Products lnc., S100 Walnut Grove, San Gabriel, CA 91778, USA. UV Products Ltd., Science Park, Milton Road, Cambridge CB4 4BN, UK . Van Waters & Rogers, PO Box 6016, Cerritos, CA 90702, USA. Virtis Co. Inc., Route 208, Gardiner, NY 12S2S, USA. c/ o Damon/ 1EC (UK) Ltd., Unit 7, Lawrence Way, Brewers Hill Road, Dunstable, Beds. LU6 lBD, UK. VWR Scientific Product , PO Box 7900, San Francisco, CA 94120, USA. Wako Pure Chemicals, Dosho-Machi, Osaka, Japan. Waring Commercial, c/ o Christison Scientific Equipment Ltd., Albany Road, East Gateshead Industrial Estate, Gateshead NE8 3AT, UK. Whatman cientific Ltd., Whatman House, St Leonards Road, Maidstone, Kent ME16 OLS, UK. Worthington Biochemical Corporation, Halls Mill Road, Freehold, NJ 01728. USA. c/ o Cambridge Bioscience Ltd., 25 Signet Court, Stourbridge Common Business Centre, Swans Road, Cambridge CBS 8LA, UK.

244

Contents of Volume I 1. Synthesis and purification of RNA substrates Benoit Chabot

2. Characterization of RNA Paula J. Grabowski

3. Splicing of rnRNA precursors in mammalian

cells Jan C. Eperon and Adrian R. Krainer

4. Isolation and characterization of ribonucleo-

protein complexes Angus I. Lamond and Bnan S. Sproat

5. Analysis of ribonucleoprotein interactions Cindy L. Will, Berthold Kaster, and Reinhard Luhrmann

6. Analysis of pre-mRNA splicing in yeast Andrew Newman

Index \!any of thl' nandard me/hods for p"parallon OJ R.\ ..J Jubolation 76-8 ,chizodeme typing of proto7oa 78-9 sl!e also kinetoplast D~A ~inetopla;,t mitochondrial lra"ion 69-11. 81-5, see also mitochondria kinetoplast RNA (kRNA) ·~lation 87-8 "onhern blot anal}\is 88 occurrence 69-71 PCR amplification of partially edited mRNAs and gRNA"mRNA chimaeras

90, 92-3 trum-c;terification model 69-70, 90, 96, 101-2 tvpes of editing 69-71 electron microscopy of rRNA synthesis 143-4 electrophoreSIS, see agarose (name) gel clectrophore.>l\, formaldehyde-agarose gd electrophoresi,, paper electrophoresis. polyacrylamide urea (denaturing) gel electrophorctructure> 5' end-proc~sing of rR"-A 135-;, 148-51.

153-5

5'

end-pr~ingoftRN .-\

175. 183, 186, 188-9

enzyme ca,.:.lde model. R'A editing 69--o non-binding sequenc~ 10 grour II ribozymes 219-20, 225, 228 e'press10n of DHFR-Iiga<e fusion protein 191-~

e\lernal transcribed spacer. pre-rRNA

135-9, 142, 144. 148, 150-1, 155, 157-60 extract preparation. for RNA processing, ~ee lysolecithin e\lracts, nuclear e\tracts, post-nuclear ~upernatant, po>t-polysomal. ~upcrnatam, processing e\lract'; see ulsu Volume I

formaldehyde-agaro'e gel ele.:trophore'l~ 10 'onhern blotting of I.R':A 108-9. 119-~1. 124, 127-~ extract for, preparation 111-19 of in l'tlro tran~cript' 108-9, 121-6, 12S-30 messenger Rl' analysis by electrophoresis 7 -I 0 modtfication-interfercnce 15-20 RNa\e protection 10-14 formation in extracts 6-10 polyadenylation of mRl'o \ cleavage and polyadenylauon spectficlly factor, purification 26-30, 32-3 in extracts coupled to endonudeolyuc deavage 21-3

interpretation of data 24-5 method and analy\i\ ~~-4 ovenie"' :!1 predea'ed R:\A substrates 23-4 general considerauon~ 2-4 in mammalian celb 1-2 non-speci fie 24 poly(A) polymerase, purification 26-32 ~ubstrate; for 2-4, 15-16 poly(A) polymerase assay 30-2 in 3 end-processing of mR;o.; ·\ 1-2. 24 purification 27-30 separation from CPSF 27-9 polymerase chain reaction (PCR) amplificauon of partially e·dtted mRNA and gRNA:mRNA chimaeras 90,

92-3 see also Volume I polysomes as endogenous substrate feor mRNA turnover 109, 119-21 non-specilic mRNA degradation in 131 preparation II 0-11, 114- 16 riboo,omal salt wash (RS\\) from 116-17 as source of messenger RNase 112 turnover of mRNA in 12'7-8 post-nuclear supernatant mRNA turnover m 123-~· preparation 117-18 treatment with micrococcal nuclea...: 118-19 post-polysomal supernatant (5130) messenger RNases in 119 preparation 114-16 pre-mRNA, pre-rRNA, pre-tRNA, see m \'itro transcription, labelling RNA, RNA ~ubstrates

processing extracts for eduing of mRNA 9~-103 end-processing of mR"lA 4-6, 21-5 mRNA turnover 111-26 rRNA 148-55 tR:-.:A 185-91, 195-9 see also cytoplasmic eMra\'ls, lysolecithin extracts, nuclear extracts. nucleolar e'\tract. post-nuclear •upernatant. post-poly~omal supernatant, and Volume I psoralen, cro~s-hnl..ing of ~n RI'.As and rR:"\-\ 142-3 pyrophmphatase>, in cap analysts 39, 52-6

rate zonal centrifugation, analysis of rRNAprotein associations 15 7-9 reticuloc~te lysate, mRNA mmover in 113, 125-6

251

index re' er-;e 'PI icing of group II intron~ 226-8 ribosomal RN ,-\ pro.:e"ing in ntro anal}m b} cro~\-linling

guanyltransfera>e (capping cnz~ me) 31--8, ~5. 47-b Iigassing factors in 3 end-proces>ing of mR:-ductiun \lillig.m. G {Edt 0·19·963296·0 £30.00 Sparalbound hardback Paperback 0-19·963295-2 £18.50 Protein Targeting Magee, A .I & Wileman. T )Erbi Sparalbound hardback 0-19·963206-5 £32.50 Paperback 0·19·963210·3 £22.50 Diagnostic Molecular Pathology: Volume II: Cell and Tis~uP Genotyping llerrinHtun. C.S. & \.ILGe. I O'U !Eds) £30.00 0-19-963239-1 Spaalbound hardback £19.50 0-19-963238-3 Paperback Neuronal ~11 Lin~ \\'nod , I~ !Edt £32.50 Spualbound hardback D-19-963346·0 £22.50 Paperback D-19-963345·2

®

The Practical Approach Series SERIES EDITORS: D. RICKWOOD and B. D. HAMES In recent years, the importance of RNA processing in the regulation of eukaryotic gene expression has become abundantly clear and so, not surprisingly, it is now an area of intense research activity. These two volumes give detailed practical guidance on all major aspects of this subject. Step-by-step practical protocols from leading laboratories are presented for studies of the termination and end-processing, capping, methylation, splicing, and editing of mRNA as well as studies of mRNA stability and processing of rRNA and tRNA. The reader is led through all the key steps required for a successful experimental investigation. This includes full descriptions of the synthesis and purification of RNA substrates for in vitro work, the characterization of specific RNAs , and the isolation and analysis of ribonucleoprotein complexes.

RNA Processing: A Practical Approach. Volumes I and II, are invaluable laboratory companions for researchers working on mRNA, rRNA. or tRNA gene expression. Their lucid explanatory style and comprehensive coverage of all the key techniques for analysing RNA processing will be welcomed by both experienced researchers and workers embarking on such studies for the first time. Volume I 0 19 963344 4 spiral hardback 0 19 963343 6 paperback Two volume set 0 19 963473 4 spiral hardback 0 19 963472 6 paperback

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