Front Cover 1
RNA Turnover in Eukaryotes: Analysis of Specialized and Quality Control RNA Decay Pathways 2
Copyright Page 5
Contents 6
Contributors 12
Preface 18
Volumes in Series 20
Section I: Analysis of Specialized mRNA Decay Pathways 46
Chapter 1: Methods to Study No-Go mRNA Decay in Saccharomyces Cerevisiae 48
1. Introduction 49
2. Design and Construction of an NGD Substrate mRNA 52
3. Methods Used to Assay Degradation Characteristics of NGD Substrates 56
4. Conclusion 63
Acknowledgments 63
References 64
Chapter 2: 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
3. Expression of a Dominant Negative Stem-Loop Binding Protein 78
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
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
6. Summary and Conclusions 89
References 89
Chapter 3: 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
9. Concluding Remarks 113
Acknowledgments 114
References 114
Chapter 4: 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
6. Northern Blot Analysis and Transcript Quantitation 131
7. Utility of the S. pombe zfs1 Model 134
Acknowledgments 137
References 137
Chapter 5: In Vivo Analysis of the Decay of Transcripts Generated by Cytoplasmic RNA Viruses 142
1. Introduction 143
2. Collecting RNA Samples 144
3. Viral RNA Decay Systems 146
4. Analysis of Viral RNA Decay 148
5. Analysis of the 3prime End of Viral RNA 156
6. Isolation of Small RNAs 162
7. Concluding Remarks 164
Acknowledgments 165
References 165
Section II: Nonsense-Mediated MRNA Decay (How Do You Study NMD What Defines An NMD Target)
Chapter 6: Qualitative and Quantitative Assessment of the Activity of the Yeast Nonsense-Mediated mRNA Decay Pathway 172
1. Introduction 173
2. Methods and Discussion 174
3. Summary 190
Acknowledgment 191
References 191
Chapter 7: 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
4. Protocol: Genetic Screen for Novel NMD Factors 202
5. Validation Strategy 207
Acknowledgments 207
References 208
Chapter 8: 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
Chapter 9: Studying Nonsense-Mediated mRNA Decay in Mammalian Cells 222
1. Introduction 223
2. Criteria for Nonsense-Mediated mRNA Decay in Mammalian Cells 224
3. Methods Used to Study NMD in Cultured Mammalian Cells 227
Acknowledgments 242
References 242
Section III: Analysis of Nuclear MRNA Decay and Non-MRNA Decay 248
Chapter 10: Estimating Nuclear mRNA Decay in Saccharomyces Cerevisiae 250
1. Introduction 251
2. Ways to Estimate Nuclear mRNA Decay 251
3. Experimental System 252
4. Protocols 259
References 263
Chapter 11: 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
4. Analysis of the Levels of Functional tRNA in Vivo 271
5. Characterization of the Loss of tRNA 278
6. Conclusions 280
Acknowledgments 280
References 281
Chapter 12: Analysis of Nonfunctional Ribosomal RNA Decay in Saccharomyces cerevisiae 284
1. Introduction 285
2. Methods 286
3. Conclusions 301
Acknowledgments 301
References 301
Section IV: Identifying Targets of an RNA Decay Factor 306
Chapter 13: Identifying Substrates of mRNA Decay Factors by a Combined RNA Interference and DNA Microarray Approach 308
1. Introduction 309
2. Results 314
3. Transient hUPF2 Knockdown in HeLa Cells 317
4. Isolation of Total-Cell RNA with TRIzol 324
5. Total-Cell RNA Cleanup with DNase Digestion Using Qiagen RNeasy 326
6. Conversion of Total-Cell RNA to Double-Stranded cDNA 328
7. Cleanup of Double-Stranded cDNA 330
8. cRNA Synthesis by In Vitro Transcription 331
9. Purification of Biotin-Labeled cRNA Transcripts Using Qiagen RNeasy 332
10. cRNA Fragmentation 334
11. Hybridization to HG-U133a Microarrays, Washing, and Scanning of Microarrays 335
12. Target Confirmation and Analysis 336
Acknowledgments 337
References 338
Chapter 14: Analysis of RNA-Protein 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
4. Methodology 347
5. Analyzing Known RNA-Protein Interactions 350
6. Three-Hybrid Screens to Identify RNA-Protein Interactions 351
7. The Three-Hybrid Screen: A General Protocol 354
8. Other Applications of the Three-Hybrid System 357
9. Concluding Remarks 358
Acknowledgments 358
References 358
Chapter 15: Co-Immunoprecipitation Techniques for Assessing RNA-Protein Interactions in Vivo 362
1. Introduction 363
2. In Vivo Ultraviolet Cross-Linking 366
3. Cell Mixing Experiment 372
4. RNA Immunoprecipitation 376
5. Discussion 381
6. Concluding Remarks 383
Acknowledgments 384
References 384
Section V: RNAi-Mediated mRNA Decay 388
Chapter 16: How to Define Targets for Small Guide RNAs in RNA Silencing: A Biochemical Approach 390
1. Introduction 391
2. Immunopurification of Aub-piRNA 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
Chapter 17: Extension of Endogenous Primers as a Tool to Detect Micro-RNA Targets 402
1. Introduction 403
2. Reverse Transcription in Cytoplasmic Extract 404
3. Amplification and Cloning 409
4. Conclusion and Perspectives 413
Acknowledgments 415
References 415
Chapter 18: 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
4. Quantifying the Effect of a miRNA on the Translation Efficiency and Stability of a Luciferase Reporter mRNA 425
5. Examining the Influence of a miRNA on the Deadenylation and Decay of a beta-Globin Reporter mRNA 429
6. Detecting siRNA- or miRNA-Directed Endonucleolytic Cleavage 434
7. Materials 436
Acknowledgments 437
References 437
Author Index 440
Subject Index 456
Color Plate Section 463