RNA turnover in eukaryotes : analysis of specialized and quality control RNA decay pathways /

CONTENTS 6
CONTRIBUTORS 12
PREFACE 18
ANALYSIS OF SPECIALIZED mRNA DECAY PATHWAYS 46
Methods to Study No-Go mRNA Decay in Saccharomyces cerevisiae 48
1. Introduction 49
2. Design and Construction of an NGD Substrate mRNA 52
2.1. Construction of an efficient ribosome pause site in a reporter mRNA 52
2.2. Construction of reporter constructs to assay the effect of a pause site on mRNA decay 55
2.3. Construction of reporter constructs to assess translational dependence of NGD 56
3. Methods Used to Assay Degradation Characteristics of NGD Substrates 56
3.1. Characterizing the decay pathway of an NGD substrate 58
3.2. Characterizing the endonucleolytic cleavage of NGD substrates 59
3.3. Mapping the site of endonucleolytic mRNA cleavage 62
3.4. Assays used to study decay characteristics of NGD mRNA substrates 62
4. Conclusion 63
ACKNOWLEDGMENTS 63
REFERENCES 64
Cell-Cycle Regulation of Histone mRNA Degradation in Mammalian Cells: Role of Translation and Oligouridylation 68
1. Introduction 69
2. Use of the Iron Response Protein to Study the Role of Translation in Histone mRNA Degradation 71
2.1. Preparation of HeLa cell lines stably expressing mRNAs containing IREs in the 5'-UTR 71
2.2. Regulating the translation of histone mRNAs containing the IRE in the 5'-UTR 75
2.3. Detection of changes in histone mRNA stability 76
3. Expression of a Dominant Negative Stem-Loop Binding Protein 78
3.1. Stable expression of SAVEE-SLBP in HeLa cells 78
3.2. Synchronization of HeLa cells using a double-thymidine block 78
3.3. Preparation of cell lysates and detection of SLBP 79
4. Circularization RT-PCR to Map 5' and 3' Ends of Histone mRNA In Vivo and In Vitro and to Define mRNA Degradation Intermediates 79
4.1. Synchronization of HeLa cells 79
4.2. Sample preparation for cloning the 3' end of in vitro-processed histone pre-mRNA 82
4.3. RNA preparation, decapping, and circularization 83
4.4. RT-PCR of circularized RNA to detect degradation intermediates 84
4.5. Cloning of cRT-PCR and degradation intermediates 86
5. Oligo(dA) RT-PCR to Visualize Oligo(U) Tails on Histone mRNA following Inhibition of DNA Synthesis or at the End of S Phase 87
5.1. Cell synchronization 88
5.2. Reverse transcription of oligo(U) tails on the 3' end 88
5.3. Polymerase chain reaction 88
6. Summary and Conclusions 89
REFERENCES 89
Assays of Adenylate Uridylate-Rich Element- Mediated mRNA Decay in Cells 92
1. Introduction 93
2. Reporter Gene System 95
3. Construction of the Reporter Gene-ARE Plasmid 96
4. Cell Culture and Transfection 98
5. Time Course and RNA Isolation 100
6. Quantitation of mRNA Levels 102
7. Analysis of qPCR Data for mRNA Half-Life 103
8. Messenger RNA Decay Pathways 110
8.1. Deadenylation assay by Northern blotting 111
8.2. Poly(A) tail assay by polymerase chain reaction 112
8.3. Decapping 113
9. Concluding Remarks 113
ACKNOWLEDGMENTS 114
REFERENCES 114
Evaluating the Control of mRNA Decay in Fission Yeast 118
1. Introduction 119
2. Studying mRNA Decay in Yeasts 120
3. Systems for Studying TZF Protein-Mediated mRNA Decay 122
4. Characterization of zfs1 as a Mediator of mRNA Decay 123
5. Use of the nmt Expression System to Evaluate zfs1-Mediated mRNA Decay 125
5.1. nmt 125
5.2. The nmt/arz1 gene expression construct 126
5.3. Schizosaccharomyces pombe nmt/arz1 transformants 128
5.4. Repression of arz1 transcription with thiamine 129
5.5. Total-cell RNA isolation 130
6. Northern Blot Analysis and Transcript Quantitation 131
6.1. Denaturing gel electrophoresis 131
6.2. Northern blotting 131
6.3. Northern blot analysis results 133
7. Utility of the S. pombe zfs1 Model 134
ACKNOWLEDGMENTS 137
REFERENCES 137
In Vivo Analysis of the Decay of Transcripts Generated by Cytoplasmic RNA Viruses 142
1. Introduction 143
2. Collecting RNA Samples 144
2.1. Total RNA extraction 144
2.2. Isolating free Sindbis viral RNAs from packaged genomic RNA 145
3. Viral RNA Decay Systems 146
3.1. Determining the time course postinfection to turn off viral transcription 146
3.2. Methods for turning off viral transcription 147
4. Analysis of Viral RNA Decay 148
4.1. Quantitative reverse transcription PCR 149
4.2. RNase protection assay 151
5. Analysis of the 3' End of Viral RNA 156
5.1. RNase H/Northern blotting 157
5.2. Linker ligation-mediated poly(A) tail assay 159
5.3. Circularization\u2013ligation 160
6. Isolation of Small RNAs 162
7. Concluding Remarks 164
ACKNOWLEDGMENTS 165
REFERENCES 165
NONSENSE-MEDIATED MRNA DECAY (HOW DO YOU STUDY NMD; WHAT DEFINES AN NMD TARGET) 170
Qualitative and Quantitative Assessment of the Activity of the Yeast Nonsense- Mediated mRNA Decay Pathway 172
1. Introduction 173
2. Methods and Discussion 174
2.1. Measuring the levels and integrity of nonsense- containing mRNAs 174
2.2. Plate assays for nonsense suppression by NMD-deficient cells 184
2.3. Toeprinting analysis of premature translation termination 185
3. Summary 190
ACKNOWLEDGMENT 191
REFERENCES 191
Nonsense-Mediated mRNA Decay in Caenorhabditis elegans 194
1. Introduction 195
2. Nonsense-Mediated mRNA Decay Reporter 195
3. Protocol for a Genome-Wide RNAi-Based NMD Screen 198
Materials 200
Day 1 201
Day 2 201
Days 5 and 6 202
4. Protocol: Genetic Screen for Novel NMD Factors 202
4.1. EMS mutagenesis 204
4.2. Selection of mutants 205
4.3. Mutant male generation 205
4.4. DNA preparation 206
4.5. Mapping 207
5. Validation Strategy 207
ACKNOWLEDGMENTS 207
REFERENCES 208
In Vivo Analysis of Plant Nonsense- Mediated mRNA Decay 210
1. Introduction 211
2. Introducing Test and Reference Genes into Plants or Cultured Plant Cells 211
3. Assessing mRNA Instability by Nonsense-Mediated mRNA Decay Inhibitor Treatment 212
4. Comparing the Relative Stabilities of Test and Reference mRNAs 214
5. Experiment 1 (Analysis of Endogeneous NMD Target: The Fate of At3g63340 Splicing Variants in Arabidopsis thaliana) 215
6. Experiment 2 (Recognition of Termination Codon Contexts as NMD Targets in Nicotiana benthamiana) 218
REFERENCES 220
Studying Nonsense-Mediated mRNA Decay in Mammalian Cells 222
1. Introduction 223
2. Criteria for Nonsense-Mediated mRNA Decay in Mammalian Cells 224
2.1. Rule for which nonsense codons trigger NMD 224
2.2. Exceptions to the 50- to 55-nucleotide rule 224
2.3. Mammalian-cell NMD is a consequence of nonsense codon recognition during a pioneer round of translation 225
2.4. Factor dependence of NMD in mammalian cells 226
3. Methods Used to Study NMD in Cultured Mammalian Cells 227
3.1. Expressing the putative NMD target 227
3.2. Transient cell transfections 228
3.3. Transient cell transfections using siRNA to downregulate Upf1, Upf2, or Upf3X 229
3.4. RT-PCR 231
3.5. Use of the c-fos promoter to determine the half-life of nucleus- associated and cytoplasmic mRNA 234
3.6. Use of translational inhibitors to study NMD 236
3.7. Immunoprecipitation of CBP80/20-bound and eIF4E-bound mRNA 239
ACKNOWLEDGMENTS 242
REFERENCES 242
ANALYSIS OF NUCLEAR MRNA DECAY AND NON-MRNA DECAY 248
Estimating Nuclear mRNA Decay in Saccharomyces cerevisiae 250
1. Introduction 251
2. Ways to Estimate Nuclear mRNA Decay 251
3. Experimental System 252
3.1. Nuclear decay of HSP104 RNA in THO/sub2 mutants: A case story 252
3.2. Summary and notes of caution 257
4. Protocols 259
4.1. Cell growth and temperature shifts 259
4.2. RNA isolation 259
4.3. RNase H treatment and Northern blotting using sequencing gels 260
4.4. Reverse transcription and quantitative polymerase chain reaction analysis of RNA 260
4.5. RNA fluorescence in situ hybridization 261
REFERENCES 263
Identification and Analysis of tRNAs That Are Degraded in Saccharomyces cerevisiae Due To Lack of Modifications 266
1. Introduction 267
2. Methodology 268
3. Identification of tRNA Species Reduced in Modification Mutants 268
3.1. Direct testing of likely tRNA targets 269
3.2. Microarray analysis of tRNA levels 269
3.3. Multicopy suppression of mutant phenotype 270
4. Analysis of the Levels of Functional tRNA In Vivo 271
4.1. Growth of cells for RNA isolation 271
4.2. Preparation of RNA for analysis of tRNA levels 273
4.3. Preparation of RNA under acidic conditions for analysis of aminoacylation 274
4.4. Northern blot hybridization and quantification 275
4.5. Experimental considerations 277
5. Characterization of the Loss of tRNA 278
6. Conclusions 280
ACKNOWLEDGMENTS 280
REFERENCES 281
Analysis of Nonfunctional Ribosomal RNA Decay in Saccharomyces cerevisiae 284
1. Introduction 285
2. Methods 286
2.1. Saccharomyces cerevisiae rDNA plasmids 286
2.2. Mutagenesis of rDNA plasmids 292
2.3. Assessing functionality of mutated rRNA 294
2.4. Quantitative analysis of mutant rRNA 295
2.5. Kinetic analysis of mutated rRNA by transcriptional pulse chase 297
2.6. Northern blot analysis 299
3. Conclusions 301
ACKNOWLEDGMENTS 301
REFERENCES 301
IDENTIFYING TARGETS OF AN RNA DECAY FACTOR 306
Identifying Substrates of mRNA Decay Factors by a Combined RNA Interference and DNA Microarray Approach 308
1. Introduction 309
2. Results 314
2.1. Required reagents 314
2.2. Reagent setup 316
2.3. Equipment 316
3. Transient hUPF2 Knockdown in HeLa Cells 317
3.1. Procedure 317
4. Isolation of Total-Cell RNA with TRIzol 324
4.1. Important points before beginning 324
4.2. Procedure 324
4.3. Quantitation and quality control of total-cell RNA 325
5. Total-Cell RNA Cleanup with DNase Digestion Using Qiagen RNeasy 326
5.1. Important points before starting 326
5.2. Procedure 327
5.3. Quantitation and quality control of total-cell RNA 328
6. Conversion of Total-Cell RNA to Double-Stranded cDNA 328
6.1. Important points before starting 328
6.2. Procedure 329
7. Cleanup of Double-Stranded cDNA 330
7.1. Procedure 330
8. cRNA Synthesis by In Vitro Transcription 331
8.1. Important points before starting 331
8.2. Procedure 331
9. Purification of Biotin-Labeled cRNA Transcripts Using Qiagen RNeasy 332
9.1. Important points before starting 332
9.2. Procedure 332
10. cRNA Fragmentation 334
10.1. Important points before starting 334
10.2. Procedure 334
11. Hybridization to HG-U133a Microarrays, Washing, and Scanning of Microarrays 335
11.1. Procedure 335
12. Target Confirmation and Analysis 336
ACKNOWLEDGMENTS 337
REFERENCES 338
Analysis of RNA\u2013Protein Interactions Using a Yeast Three- Hybrid System 340
1. Introduction 341
2. Principles of the Method 342
3. Key Components: RNAs, Vectors, and Strains 343
3.1. Hybrid RNAs 343
3.2. Plasmids 345
3.3. Yeast strains 347
4. Methodology 347
4.1. Assaying interactions: B-Galactosidase activity and 3-aminotriazole resistance 348
4.2. Qualitative assays 348
4.3. Quantitative assays 348
5. Analyzing Known RNA\u2013Protein Interactions 350
5.1. Assay limitations 351
6. Three-Hybrid Screens to Identify RNA\u2013 Protein Interactions 351
6.1. Types of screens 351
6.2. A priori considerations 352
7. The Three-hybrid Screen: A General Protocol 354
Step 1. Transform yeast and select for growth 354
Step 2. Assay B-galactosidase activity 354
Step 3. Isolate plasmids 355
Step 4. Test for bait dependence (autoactivation) 355
Step 5. Identify positive clones 356
Step 6. Determine binding specificity using mutant and control molecules 356
Step 7. Functional tests or additional screens 356
8. Other Applications of the Three-Hybrid System 357
8.1. RNA activators 357
8.2. Examination of RNA aptamers 357
8.3. Multiprotein complexes 357
9. Concluding Remarks 358
ACKNOWLEDGMENTS 358
REFERENCES 358
Co-Immunoprecipitation Techniques for Assessing RNA\u2013Protein Interactions In Vivo 362
1. Introduction 363
2. In Vivo Ultraviolet Cross-Linking 366
2.1. Materials and buffers 367
2.2. Procedure 368
2.3. Results 371
3. Cell Mixing Experiment 372
3.1. Materials and buffers 373
3.2. Procedure 374
3.3. Results 376
4. RNA Immunoprecipitation 376
4.1. Materials and buffers 377
4.2. Procedure 378
4.3. Results 380
5. Discussion 381
6. Concluding Remarks 383
ACKNOWLEDGMENTS 384
REFERENCES 384
RNAi-MEDIATED mRNA DECAY 388
How to Define Targets for Small Guide RNAs in RNA Silencing: A Biochemical Approach 390
1. Introduction 391
2. Immunopurification of Aub\u2013piRNA Complexes from Fly Testis Lysates 393
3. Analyzing Protein Components Present in Immunoprecipitates by Silver Staining and Western Blot Analysis 394
4. Analyzing Small RNAs Present in Immunoprecipitates by Northern Blot Analysis 394
5. Target RNAs for Small RNA-Guided Cleavage 396
6. In Vitro Target RNA Cleavage (Slicer) Assay 397
ACKNOWLEDGMENTS 398
REFERENCES 399
Extension of Endogenous Primers as a Tool to Detect Micro- RNA Targets 402
1. Introduction 403
2. Reverse Transcription in Cytoplasmic Extract 404
2.1. Cytoplasmic extract preparation 405
2.2. Reverse transcription reaction # 1 406
2.3. Reverse transcription reaction # 2 407
3. Amplification and Cloning 409
3.1. Pre-PCR with gene-specific primer 410
3.2. Poly(A) tailing 410
3.3. Final amplification 411
3.4. Cloning 413
4. Conclusion and Perspectives 413
ACKNOWLEDGMENTS 415
REFERENCES 415
Examining the Influence of MicroRNAs on Translation Efficiency and on mRNA Deadenylation and Decay 418
1. Introduction 419
2. Predicting miRNA-Responsive Elements in mRNA by Sequence Analysis 420
3. Using a Luciferase Reporter to Examine miRE Function 420
3.1. Luciferase reporter 421
3.2. Ectopic production of a miRNA in cells where it is normally absent 421
3.3. Examining miRE function in cells where a complementary miRNA is produced naturally 423
4. Quantifying the Effect of a miRNA on the Translation Efficiency and Stability of a Luciferase Reporter mRNA 425
Method 1: Transfection of cells with plasmids encoding a reporter and a miRNA 425
Method 2: Assaying luciferase reporter activity in transfected cells 426
Method 3: Extraction of cytoplasmic RNA from transfected cells 428
Method 4: Quantifying luciferase reporter mRNA levels by Northern blotting 428
5. Examining the Influence of a miRNA on the Deadenylation and Decay of a b- Globin Reporter mRNA 429
Method 5: Monitoring the effect of a miRNA on the rate of mRNA decay 432
Method 6: Monitoring the effect of a miRNA on the rate at which mRNA is deadenylated 433
6. Detecting siRNA- or miRNA-Directed Endonucleolytic Cleavage 434
Method 7: Using RLM-RACE to detect endonucleolytic cleavage mediated by a perfectly complementary si/ miRNA 435
7. Materials 436
7.1. Cell culture media 436
7.2. Buffers and solutions 436
ACKNOWLEDGMENTS 437
REFERENCES 437
Author Index 440
Subject Index 456