RNA Turnover in Eukaryotes: Analysis of Specialized and Quality Control RNA Decay Pathways

Cover Page......Page 1
Series Editors......Page 2
Methods in Enzymology......Page 0
Copyright
......Page 3
Contributors......Page 4
Preface......Page 9
Methods in Enzymology
......Page 11
Methods to Study No-Go mRNA Decay in Saccharomyces cerevisiae......Page 37
Introduction......Page 38
Construction of an efficient ribosome pause site in a reporter mRNA......Page 41
Construction of reporter constructs to assay the effect of a pause site on mRNA decay......Page 44
Methods Used to Assay Degradation Characteristics of NGD Substrates......Page 45
Characterizing the decay pathway of an NGD substrate......Page 47
Characterizing the endonucleolytic cleavage of NGD substrates......Page 48
Assays used to study decay characteristics of NGD mRNA substrates......Page 51
Acknowledgments......Page 52
References......Page 53
Cell-Cycle Regulation of Histone mRNA Degradation in Mammalian Cells: Role of Translation and Oligouridylation......Page 56
Introduction......Page 57
DNA constructs......Page 59
Creation of HeLa cell lines that stably express mouse histone H2a genes containing either a wild-type IRE or a mutated IRE in the 5'-UTR......Page 62
Regulating the translation of histone mRNAs containing the IRE in the 5'-UTR......Page 63
Detection of changes in histone mRNA stability......Page 64
Hybridization......Page 65
Synchronization of HeLa cells using a double-thymidine block......Page 66
Synchronization of HeLa cells......Page 67
Sample preparation for cloning the 3' end of in vitro-processed histone pre-mRNA......Page 70
Decapping......Page 71
RT-PCR of circularized RNA to detect degradation intermediates......Page 72
T/A cloning of cRT-PCR reactions......Page 74
Oligo(dA) RT-PCR to Visualize Oligo(U) Tails on Histone mRNA following Inhibition of DNA Synthesis or at the End of S Phase......Page 75
Polymerase chain reaction......Page 76
References......Page 77
Assays of Adenylate Uridylate-Rich Element-Mediated mRNA Decay in Cells......Page 79
Introduction......Page 80
Reporter Gene System......Page 82
Construction of the Reporter Gene-ARE Plasmid......Page 83
Procedure......Page 84
Cell Culture and Transfection......Page 85
Procedure......Page 86
Procedure......Page 87
Procedure......Page 89
Analysis of qPCR Data for mRNA Half-Life......Page 90
Procedure......Page 93
Deadenylation assay by Northern blotting......Page 97
Procedure......Page 98
Procedure......Page 99
Concluding Remarks......Page 100
References......Page 101
Evaluating the Control of mRNA Decay in Fission Yeast......Page 104
Introduction......Page 105
Studying mRNA Decay in Yeasts......Page 106
Systems for Studying TZF Protein-Mediated mRNA Decay......Page 108
Characterization of zfs1 as a Mediator of mRNA Decay......Page 109
nmt......Page 111
The nmt/arz1 gene expression construct......Page 112
Schizosaccharomyces pombe nmt/arz1 transformants......Page 114
Repression of arz1 transcription with thiamine......Page 115
Total-cell RNA isolation......Page 116
Northern blotting......Page 117
Northern blot analysis results......Page 119
Utility of the S. pombe zfs1 Model......Page 120
References......Page 123
In Vivo Analysis of the Decay of Transcripts Generated by Cytoplasmic RNA Viruses......Page 127
Introduction......Page 128
Total RNA extraction......Page 129
Procedure......Page 130
Determining the time course postinfection to turn off viral transcription......Page 131
Procedure......Page 132
Analysis of Viral RNA Decay......Page 133
Quantitative reverse transcription PCR......Page 134
Procedures......Page 135
RNase protection assay......Page 136
Procedure......Page 137
Analysis of the 3' End of Viral RNA......Page 141
Procedure......Page 142
Procedure......Page 144
Circularization-ligation......Page 145
Procedure......Page 146
Isolation of small RNAs from total RNA......Page 147
Selecting and visualizing small RNAs......Page 148
Concluding Remarks......Page 149
References......Page 150
Qualitative and Quantitative Assessment of the Activity of the Yeast Nonsense-Mediated mRNA Decay Pathway......Page 154
Introduction......Page 155
Reagents and buffers......Page 156
Yeast strains and growth conditions......Page 157
Analysis of mRNA steady-state levels......Page 158
Determination of the 5'-cap status......Page 160
Defining mRNA 5' ends......Page 162
Measurement of the size of mRNA 3'-poly(A) tails......Page 163
Polyacrylamide northern blotting......Page 164
Genome-wide profiling of transcript levels......Page 165
Media......Page 166
Toeprinting analysis of premature translation termination......Page 167
Growth of culture and cell lysis......Page 168
Chromatography and nuclease treatment of extracts......Page 169
Sequencing reactions......Page 170
Application of the methodology......Page 171
Summary......Page 172
References......Page 173
Nonsense-Mediated mRNA Decay in Caenorhabditis elegans......Page 175
Nonsense-Mediated mRNA Decay Reporter......Page 176
Protocol for a Genome-Wide RNAi-Based NMD Screen......Page 179
Materials......Page 181
Day 2......Page 182
Protocol: Genetic Screen for Novel NMD Factors......Page 183
EMS mutagenesis......Page 185
Mutant male generation......Page 186
DNA preparation......Page 187
Acknowledgments......Page 188
References......Page 189
In Vivo Analysis of Plant Nonsense-Mediated mRNA Decay......Page 191
Introducing Test and Reference Genes into Plants or Cultured Plant Cells......Page 192
Assessing mRNA Instability by Nonsense-Mediated mRNA Decay Inhibitor Treatment......Page 193
Comparing the Relative Stabilities of Test and Reference mRNAs......Page 195
Experiment 1 (Analysis of Endogeneous NMD Target: The Fate of At3g63340 Splicing Variants in Arabidopsis thaliana)......Page 196
Experiment 2 (Recognition of Termination Codon Contexts as NMD Targets in Nicotiana benthamiana)......Page 199
References......Page 201
Studying Nonsense-Mediated mRNA Decay in Mammalian Cells......Page 203
Introduction......Page 204
Exceptions to the 50- to 55-nucleotide rule......Page 205
Mammalian-cell NMD is a consequence of nonsense codon recognition during a pioneer round of translation......Page 206
Factor dependence of NMD in mammalian cells......Page 207
Expressing the putative NMD target......Page 208
Transient cell transfections......Page 209
Transient cell transfections using siRNA to downregulate Upf1, Upf2, or Upf3X......Page 210
RT-PCR......Page 212
Reverse transcriptase (RT) cocktail for cDNA synthesis......Page 214
Use of the c-fos promoter to determine the half-life of nucleus-associated and cytoplasmic mRNA......Page 215
Use of translational inhibitors to study NMD......Page 217
Immunoprecipitation of CBP80/20-bound and eIF4E-bound mRNA......Page 220
References......Page 223
Estimating Nuclear mRNA Decay in Saccharomyces cerevisiae......Page 228
Ways to Estimate Nuclear mRNA Decay......Page 229
Nuclear decay of HSP104 RNA in THO/sub2 mutants: A case story......Page 230
Summary and notes of caution......Page 235
RNA isolation......Page 237
Reverse transcription and quantitative polymerase chain reaction analysis of RNA......Page 238
Slide preparation and spheroblasting......Page 239
Hybridization......Page 240
References......Page 241
Identification and Analysis of tRNAs That Are Degraded in Saccharomyces cerevisiae Due To Lack of Modifications......Page 243
Introduction......Page 244
Identification of tRNA Species Reduced in Modification Mutants......Page 245
Microarray analysis of tRNA levels......Page 246
Multicopy suppression of mutant phenotype......Page 247
Growth of cells for RNA isolation......Page 248
Preparation of RNA for analysis of tRNA levels......Page 250
Preparation of RNA under acidic conditions for analysis of aminoacylation......Page 251
Northern blot hybridization and quantification......Page 252
Experimental considerations......Page 254
Characterization of the Loss of tRNA......Page 255
Acknowledgments......Page 257
References......Page 258
Analysis of Nonfunctional Ribosomal RNA Decay in Saccharomyces cerevisiae......Page 260
Introduction......Page 261
Saccharomyces cerevisiae rDNA plasmids......Page 262
Mutagenesis of rDNA plasmids......Page 268
Assessing functionality of mutated rRNA......Page 270
Quantitative analysis of mutant rRNA......Page 271
Kinetic analysis of mutated rRNA by transcriptional pulse chase......Page 273
Northern blot analysis......Page 275
References......Page 277
Identifying Substrates of mRNA Decay Factors by a Combined RNA Interference and DNA Microarray Approach......Page 281
Introduction......Page 282
Chemicals......Page 287
Miscellaneous......Page 288
Equipment......Page 289
Identification of a functional siRNA sequence by transient siRNA transfection......Page 290
Generation of a hUPF2 shRNA plasmid for stable protein knockdown......Page 293
Cloning a double-stranded
DNA oligonucleotide into pSUPER......Page 294
Inducible protein knockdown and retroviral delivery of shRNAs......Page 295
Stable transfection of HeLa cells with pSUPER plasmids......Page 296
Procedure......Page 297
Quantitation and quality control of total-cell RNA......Page 298
Important points before starting......Page 299
Procedure......Page 300
Important points before starting......Page 301
First-strand cDNA synthesis......Page 302
Phase-lock gel tubes phenol/chloroform extraction......Page 303
Procedure......Page 304
Purification of biotin-labeled cRNA transcripts......Page 305
Quantification of cRNA......Page 306
Procedure......Page 307
Procedure......Page 308
Target Confirmation and Analysis......Page 309
Acknowledgments......Page 310
References......Page 311
Analysis of RNA-Protein Interactions Using a Yeast Three-Hybrid System......Page 313
Introduction......Page 314
Principles of the Method......Page 315
Hybrid RNAs......Page 316
p3HR2 (Stumpf et al., 2008)......Page 318
Plasmids encoding the activation domain fusion......Page 319
Methodology......Page 320
Relationship between reporter gene activity and affinity......Page 321
Protocol for quantitative beta-galactosidase assays......Page 322
Analyzing Known RNA-Protein Interactions......Page 323
Types of screens......Page 324
Stringency......Page 325
Libraries......Page 326
Step 2. Assay beta-galactosidase activity......Page 327
Step 4. Test for bait dependence (autoactivation)......Page 328
Step 7. Functional tests or additional screens......Page 329
Multiprotein complexes......Page 330
References......Page 331
Co-Immunoprecipitation Techniques for Assessing RNA-Protein Interactions In Vivo......Page 334
Introduction......Page 335
In Vivo Ultraviolet Cross-Linking......Page 338
Materials and buffers......Page 339
Procedure......Page 340
Results......Page 343
Cell Mixing Experiment......Page 344
Materials and buffers......Page 345
Procedure......Page 346
RNA Immunoprecipitation......Page 348
Materials and buffers......Page 349
Procedure......Page 350
Results......Page 352
Discussion......Page 353
Concluding Remarks......Page 355
References......Page 356
How to Define Targets for Small Guide RNAs in RNA Silencing: A Biochemical Approach......Page 360
Introduction......Page 361
Immunopurification of Aub-piRNA Complexes from Fly Testis Lysates......Page 363
Analyzing Small RNAs Present in Immunoprecipitates by Northern Blot Analysis......Page 364
Target RNAs for Small RNA-Guided Cleavage......Page 366
In Vitro Target RNA Cleavage (Slicer) Assay......Page 367
Acknowledgments......Page 368
References......Page 369
Extension of Endogenous Primers as a Tool to Detect Micro-RNA Targets......Page 371
Introduction......Page 372
Reverse Transcription in Cytoplasmic Extract......Page 373
Cytoplasmic extract preparation......Page 374
Reverse transcription reaction #1......Page 375
Reverse transcription reaction #2......Page 376
Amplification and Cloning......Page 378
Poly(A) tailing......Page 379
Final amplification......Page 380
Conclusion and Perspectives......Page 382
References......Page 384
Examining the Influence of MicroRNAs on Translation Efficiency and on mRNA Deadenylation and Decay......Page 386
Introduction......Page 387
Using a Luciferase Reporter to Examine miRE Function......Page 388
Ectopic production of a miRNA in cells where it is normally absent......Page 389
Examining miRE function in cells where a complementary miRNA is produced naturally......Page 391
Method 1: Transfection of cells with plasmids encoding a reporter and a miRNA......Page 393
Method 2: Assaying luciferase reporter activity in transfected cells......Page 394
Method 4: Quantifying luciferase reporter mRNA levels by Northern blotting......Page 396
Examining the Influence of a miRNA on the Deadenylation and Decay of a beta-Globin Reporter mRNA......Page 397
Method 5: Monitoring the effect of a miRNA on the rate of mRNA decay......Page 400
Method 6: Monitoring the effect of a miRNA on the rate at which mRNA is deadenylated......Page 401
Detecting siRNA- or miRNA-Directed Endonucleolytic Cleavage......Page 402
Method 7: Using RLM-RACE to detect endonucleolytic cleavage mediated by a perfectly complementary si/miRNA......Page 403
Buffers and solutions......Page 404
References......Page 405
Author Index......Page 407
E......Page 423
M......Page 424
N......Page 425
R......Page 426
T......Page 427
Z......Page 428