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

Methods in Enzymology......Page 0
Copyright\r......Page 2
Contributors......Page 3
Methods in Enzymology\r......Page 10
Methods to Study No-Go mRNA Decay in Saccharomyces cerevisiae......Page 36
Introduction......Page 37
Construction of an efficient ribosome pause site in a reporter mRNA......Page 40
Construction of reporter constructs to assay the effect of a pause site on mRNA decay......Page 43
Methods Used to Assay Degradation Characteristics of NGD Substrates......Page 44
Characterizing the decay pathway of an NGD substrate......Page 46
Characterizing the endonucleolytic cleavage of NGD substrates......Page 47
Assays used to study decay characteristics of NGD mRNA substrates......Page 50
Acknowledgments......Page 51
References......Page 52
Cell-Cycle Regulation of Histone mRNA Degradation in Mammalian Cells: Role of Translation and Oligouridylation......Page 55
Introduction......Page 56
DNA constructs......Page 58
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 61
Regulating the translation of histone mRNAs containing the IRE in the 5\'-UTR......Page 62
Detection of changes in histone mRNA stability......Page 63
Hybridization......Page 64
Synchronization of HeLa cells using a double-thymidine block......Page 65
Synchronization of HeLa cells......Page 66
Sample preparation for cloning the 3\' end of in vitro-processed histone pre-mRNA......Page 69
Decapping......Page 70
RT-PCR of circularized RNA to detect degradation intermediates......Page 71
T/A cloning of cRT-PCR reactions......Page 73
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 74
Polymerase chain reaction......Page 75
References......Page 76
Assays of Adenylate Uridylate-Rich Element-Mediated mRNA Decay in Cells......Page 78
Introduction......Page 79
Reporter Gene System......Page 81
Construction of the Reporter Gene-ARE Plasmid......Page 82
Procedure......Page 83
Cell Culture and Transfection......Page 84
Procedure......Page 85
Procedure......Page 86
Procedure......Page 88
Analysis of qPCR Data for mRNA Half-Life......Page 89
Procedure......Page 92
Deadenylation assay by Northern blotting......Page 96
Procedure......Page 97
Procedure......Page 98
Concluding Remarks......Page 99
References......Page 100
Evaluating the Control of mRNA Decay in Fission Yeast......Page 103
Introduction......Page 104
Studying mRNA Decay in Yeasts......Page 105
Systems for Studying TZF Protein-Mediated mRNA Decay......Page 107
Characterization of zfs1 as a Mediator of mRNA Decay......Page 108
nmt......Page 110
The nmt/arz1 gene expression construct......Page 111
Schizosaccharomyces pombe nmt/arz1 transformants......Page 113
Repression of arz1 transcription with thiamine......Page 114
Total-cell RNA isolation......Page 115
Northern blotting......Page 116
Northern blot analysis results......Page 118
Utility of the S. pombe zfs1 Model......Page 119
References......Page 122
In Vivo Analysis of the Decay of Transcripts Generated by Cytoplasmic RNA Viruses......Page 126
Introduction......Page 127
Total RNA extraction......Page 128
Procedure......Page 129
Determining the time course postinfection to turn off viral transcription......Page 130
Procedure......Page 131
Analysis of Viral RNA Decay......Page 132
Quantitative reverse transcription PCR......Page 133
Procedures......Page 134
RNase protection assay......Page 135
Procedure......Page 136
Analysis of the 3\' End of Viral RNA......Page 140
Procedure......Page 141
Procedure......Page 143
Circularization-ligation......Page 144
Procedure......Page 145
Isolation of small RNAs from total RNA......Page 146
Selecting and visualizing small RNAs......Page 147
Concluding Remarks......Page 148
References......Page 149
Qualitative and Quantitative Assessment of the Activity of the Yeast Nonsense-Mediated mRNA Decay Pathway......Page 153
Introduction......Page 154
Reagents and buffers......Page 155
Yeast strains and growth conditions......Page 156
Analysis of mRNA steady-state levels......Page 157
Determination of the 5\'-cap status......Page 159
Defining mRNA 5\' ends......Page 161
Measurement of the size of mRNA 3\'-poly(A) tails......Page 162
Polyacrylamide northern blotting......Page 163
Genome-wide profiling of transcript levels......Page 164
Media......Page 165
Toeprinting analysis of premature translation termination......Page 166
Growth of culture and cell lysis......Page 167
Chromatography and nuclease treatment of extracts......Page 168
Sequencing reactions......Page 169
Application of the methodology......Page 170
Summary......Page 171
References......Page 172
Nonsense-Mediated mRNA Decay in Caenorhabditis elegans......Page 174
Nonsense-Mediated mRNA Decay Reporter......Page 175
Protocol for a Genome-Wide RNAi-Based NMD Screen......Page 178
Materials......Page 180
Day 2......Page 181
Protocol: Genetic Screen for Novel NMD Factors......Page 182
EMS mutagenesis......Page 184
Mutant male generation......Page 185
DNA preparation......Page 186
Acknowledgments......Page 187
References......Page 188
In Vivo Analysis of Plant Nonsense-Mediated mRNA Decay......Page 190
Introducing Test and Reference Genes into Plants or Cultured Plant Cells......Page 191
Assessing mRNA Instability by Nonsense-Mediated mRNA Decay Inhibitor Treatment......Page 192
Comparing the Relative Stabilities of Test and Reference mRNAs......Page 194
Experiment 1 (Analysis of Endogeneous NMD Target: The Fate of At3g63340 Splicing Variants in Arabidopsis thaliana)......Page 195
Experiment 2 (Recognition of Termination Codon Contexts as NMD Targets in Nicotiana benthamiana)......Page 198
References......Page 200
Studying Nonsense-Mediated mRNA Decay in Mammalian Cells......Page 202
Introduction......Page 203
Exceptions to the 50- to 55-nucleotide rule......Page 204
Mammalian-cell NMD is a consequence of nonsense codon recognition during a pioneer round of translation......Page 205
Factor dependence of NMD in mammalian cells......Page 206
Expressing the putative NMD target......Page 207
Transient cell transfections......Page 208
Transient cell transfections using siRNA to downregulate Upf1, Upf2, or Upf3X......Page 209
RT-PCR......Page 211
Reverse transcriptase (RT) cocktail for cDNA synthesis......Page 213
Use of the c-fos promoter to determine the half-life of nucleus-associated and cytoplasmic mRNA......Page 214
Use of translational inhibitors to study NMD......Page 216
Immunoprecipitation of CBP80/20-bound and eIF4E-bound mRNA......Page 219
References......Page 222
Estimating Nuclear mRNA Decay in Saccharomyces cerevisiae......Page 227
Ways to Estimate Nuclear mRNA Decay......Page 228
Nuclear decay of HSP104 RNA in THO/sub2 mutants: A case story......Page 229
Summary and notes of caution......Page 234
RNA isolation......Page 236
Reverse transcription and quantitative polymerase chain reaction analysis of RNA......Page 237
Slide preparation and spheroblasting......Page 238
Hybridization......Page 239
References......Page 240
Identification and Analysis of tRNAs That Are Degraded in Saccharomyces cerevisiae Due To Lack of Modifications......Page 242
Introduction......Page 243
Identification of tRNA Species Reduced in Modification Mutants......Page 244
Microarray analysis of tRNA levels......Page 245
Multicopy suppression of mutant phenotype......Page 246
Growth of cells for RNA isolation......Page 247
Preparation of RNA for analysis of tRNA levels......Page 249
Preparation of RNA under acidic conditions for analysis of aminoacylation......Page 250
Northern blot hybridization and quantification......Page 251
Experimental considerations......Page 253
Characterization of the Loss of tRNA......Page 254
Acknowledgments......Page 256
References......Page 257
Analysis of Nonfunctional Ribosomal RNA Decay in Saccharomyces cerevisiae......Page 259
Introduction......Page 260
Saccharomyces cerevisiae rDNA plasmids......Page 261
Mutagenesis of rDNA plasmids......Page 267
Assessing functionality of mutated rRNA......Page 269
Quantitative analysis of mutant rRNA......Page 270
Kinetic analysis of mutated rRNA by transcriptional pulse chase......Page 272
Northern blot analysis......Page 274
References......Page 276
Identifying Substrates of mRNA Decay Factors by a Combined RNA Interference and DNA Microarray Approach......Page 280
Introduction......Page 281
Chemicals......Page 286
Miscellaneous......Page 287
Equipment......Page 288
Identification of a functional siRNA sequence by transient siRNA transfection......Page 289
Generation of a hUPF2 shRNA plasmid for stable protein knockdown......Page 292
Cloning a double-stranded \rDNA oligonucleotide into pSUPER......Page 293
Inducible protein knockdown and retroviral delivery of shRNAs......Page 294
Stable transfection of HeLa cells with pSUPER plasmids......Page 295
Procedure......Page 296
Quantitation and quality control of total-cell RNA......Page 297
Important points before starting......Page 298
Procedure......Page 299
Important points before starting......Page 300
First-strand cDNA synthesis......Page 301
Phase-lock gel tubes phenol/chloroform extraction......Page 302
Procedure......Page 303
Purification of biotin-labeled cRNA transcripts......Page 304
Quantification of cRNA......Page 305
Procedure......Page 306
Procedure......Page 307
Target Confirmation and Analysis......Page 308
Acknowledgments......Page 309
References......Page 310
Analysis of RNA-Protein Interactions Using a Yeast Three-Hybrid System......Page 312
Introduction......Page 313
Principles of the Method......Page 314
Hybrid RNAs......Page 315
p3HR2 (Stumpf et al., 2008)......Page 317
Plasmids encoding the activation domain fusion......Page 318
Methodology......Page 319
Relationship between reporter gene activity and affinity......Page 320
Protocol for quantitative beta-galactosidase assays......Page 321
Analyzing Known RNA-Protein Interactions......Page 322
Types of screens......Page 323
Stringency......Page 324
Libraries......Page 325
Step 2. Assay beta-galactosidase activity......Page 326
Step 4. Test for bait dependence (autoactivation)......Page 327
Step 7. Functional tests or additional screens......Page 328
Multiprotein complexes......Page 329
References......Page 330
Co-Immunoprecipitation Techniques for Assessing RNA-Protein Interactions In Vivo......Page 333
Introduction......Page 334
In Vivo Ultraviolet Cross-Linking......Page 337
Materials and buffers......Page 338
Procedure......Page 339
Results......Page 342
Cell Mixing Experiment......Page 343
Materials and buffers......Page 344
Procedure......Page 345
RNA Immunoprecipitation......Page 347
Materials and buffers......Page 348
Procedure......Page 349
Results......Page 351
Discussion......Page 352
Concluding Remarks......Page 354
References......Page 355
How to Define Targets for Small Guide RNAs in RNA Silencing: A Biochemical Approach......Page 359
Introduction......Page 360
Immunopurification of Aub-piRNA Complexes from Fly Testis Lysates......Page 362
Analyzing Small RNAs Present in Immunoprecipitates by Northern Blot Analysis......Page 363
Target RNAs for Small RNA-Guided Cleavage......Page 365
In Vitro Target RNA Cleavage (Slicer) Assay......Page 366
Acknowledgments......Page 367
References......Page 368
Extension of Endogenous Primers as a Tool to Detect Micro-RNA Targets......Page 370
Introduction......Page 371
Reverse Transcription in Cytoplasmic Extract......Page 372
Cytoplasmic extract preparation......Page 373
Reverse transcription reaction #1......Page 374
Reverse transcription reaction #2......Page 375
Amplification and Cloning......Page 377
Poly(A) tailing......Page 378
Final amplification......Page 379
Conclusion and Perspectives......Page 381
References......Page 383
Examining the Influence of MicroRNAs on Translation Efficiency and on mRNA Deadenylation and Decay......Page 385
Introduction......Page 386
Using a Luciferase Reporter to Examine miRE Function......Page 387
Ectopic production of a miRNA in cells where it is normally absent......Page 388
Examining miRE function in cells where a complementary miRNA is produced naturally......Page 390
Method 1: Transfection of cells with plasmids encoding a reporter and a miRNA......Page 392
Method 2: Assaying luciferase reporter activity in transfected cells......Page 393
Method 4: Quantifying luciferase reporter mRNA levels by Northern blotting......Page 395
Examining the Influence of a miRNA on the Deadenylation and Decay of a beta-Globin Reporter mRNA......Page 396
Method 5: Monitoring the effect of a miRNA on the rate of mRNA decay......Page 399
Method 6: Monitoring the effect of a miRNA on the rate at which mRNA is deadenylated......Page 400
Detecting siRNA- or miRNA-Directed Endonucleolytic Cleavage......Page 401
Method 7: Using RLM-RACE to detect endonucleolytic cleavage mediated by a perfectly complementary si/miRNA......Page 402
Buffers and solutions......Page 403
References......Page 404
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