RNA Turnover in Eukaryotes: Nucleases, Pathways and Analysis of mRNA Decay

Methods in Enzymology......Page 0
Copyright Page......Page 2
Contributors......Page 3
Preface......Page 9
Methods in Enzymology......Page 11
Analysis of mRNA Decapping......Page 37
Introduction......Page 38
Measuring Decapping Activities of Recombinant and Endogenous Dcp2......Page 40
In vitro Transcription......Page 41
Gel purification of the cap-labeled RNA......Page 42
Measuring Dcp2 decapping activity......Page 43
Preparation of recombinant flag-tagged hDcp2 expressed in human cells......Page 44
Preparation of endogenous hDcp2 (P50 cytoplasmic fraction) for decapping assays......Page 45
In vitro Dcp2 decapping assay......Page 46
Presence of a copurifying bacterial pyrophosphatase activity......Page 48
Measuring DcpS Activity......Page 49
Generation of total-cell extract to detect endogenous DcpS activity......Page 50
Migration of Cap Analogs with Various Thin-Layer Chromatography Running Buffers......Page 51
Preparation of m7GTP......Page 52
References......Page 53
A Kinetic Assay to Monitor RNA Decapping Under Single-Turnover Conditions......Page 56
Introduction......Page 57
Kinetic Equations......Page 58
Cap-labeled RNA preparation......Page 61
Substrate characterization......Page 62
Overview......Page 63
TLC analysis......Page 65
Curve fitting......Page 66
Metal activation......Page 67
Summary......Page 69
Buffers......Page 70
References......Page 71
Purification and Analysis of the Decapping Activator Lsm1p-7p-Pat1p Complex from Yeast......Page 74
Introduction......Page 75
Purification of the Lsm1p-7p-Pat1p Complex......Page 76
Buffers (prepared with the stock solutions listed above)......Page 78
Preclearing of lysate......Page 79
Ni-NTA matrix binding and elution......Page 80
Analysis of RNA Binding by the Lsm1p-7p-Pat1p Complex......Page 83
References......Page 86
Reconstitution of Recombinant Human LSm Complexes for Biochemical, Biophysical, and Cell Biological Studies......Page 89
Introduction......Page 90
Cloning......Page 93
Protein Expression and Purification......Page 97
LSm Complex Reconstitution......Page 100
LSm Complex Functional Assays......Page 102
References......Page 104
Regulated Deadenylation in Vitro......Page 107
Introduction......Page 108
Advantages......Page 110
A brief history......Page 111
Optimizing deadenylation conditions......Page 112
RNA substrates......Page 114
5\'-End labeling......Page 115
Protocol: 5\'-labeling of synthetic substrate RNA......Page 116
Purified components......Page 117
Harvest cells......Page 118
Exchange buffer......Page 120
Control reactions......Page 121
Analyze products on a denaturing polyacrylamide gel......Page 123
Analyze purified regulator......Page 124
Test activity of purified regulator......Page 125
Interpret data......Page 126
Assay-regulated deadenylation in vitro......Page 127
Interpret data......Page 129
Regulator concentration......Page 130
References......Page 131
Introduction......Page 137
Preparation of Drosophila Embryo Extracts......Page 140
Preparation of Substrate RNA......Page 142
Deadenylation Assay with Drosophila Embryo Extracts......Page 143
Characterization of Sequence-Dependent Deadenylation in Drosophila Embryo Extracts......Page 145
References......Page 146
Measuring CPEB-Mediated Cytoplasmic Polyadenylation-Deadenylation in Xenopus laevis Oocytes and Egg Extracts......Page 149
Introduction......Page 150
Principle......Page 151
Oocyte collection, injection, incubation, and retrieval of radiolabeled RNA probes......Page 152
Methods......Page 153
Notes......Page 155
Methods......Page 156
Principle......Page 158
Notes......Page 159
Methods......Page 160
Notes......Page 161
Methods......Page 162
Depletion of protein with specific antibody and polyadenylation assay......Page 163
Egg extract preparation......Page 164
Depletion of protein with specific antibody and polyadenylation assay......Page 165
Depletion of protein with specific interacting protein and polyadenylation assay......Page 166
References......Page 167
The Preparation and Applications of Cytoplasmic Extracts from Mammalian Cells for Studying Aspects of mRNA Decay......Page 169
Introduction......Page 170
Preparation of HeLa-Cell Cytoplasmic Extracts......Page 172
Preparation of HeLa-Cell S100 cytoplasmic extracts......Page 173
Standardization of cytoplasmic extract activity......Page 175
Protocol......Page 177
Transcription of polyadenylated and nonadenylated RNA substrates......Page 178
Production of cap-labeled RNA substrate......Page 181
Protocol......Page 182
Determining exonuclease activity......Page 183
Protocol......Page 185
Protocol......Page 186
Analysis of trans-acting factors with ultraviolet crosslinking......Page 187
Protocol......Page 188
Immunoprecipitation protocol......Page 190
Concluding Remarks......Page 191
References......Page 192
In Vitro Assays of 5\' to 3\'-Exoribonuclease Activity......Page 194
Introduction......Page 195
Purification of Xrn1......Page 197
In Vitro RNA Substrate Synthesis......Page 198
TCA-based exoribonuclease assays......Page 200
Gel-based exoribonuclease assays......Page 201
Degradation of doubly labeled RNA by Xrn1......Page 203
Degradation of 5\'- and 3\'-labeled synthetic RNAs by Xrn1......Page 206
Conclusions and Prospects......Page 207
References......Page 208
Reconstitution of RNA Exosomes from Human and Saccharomyces cerevisiae: Cloning, Expression, Purification, and Activity Assays......Page 211
Introduction......Page 212
Cloning Strategies for Recombinant Protein Expression......Page 216
Yeast RRP41/RRP45 cDNA......Page 217
Yeast RRP6 cDNA fused to SMT3 cDNA......Page 219
Human RRP4, RRP40, and CSL4 cDNA......Page 220
Expression and Purification of Yeast Exosome Proteins......Page 221
Yeast Mtr3/Rrp42......Page 222
Yeast Rrp44......Page 223
Expression and Purification of Human Exosome Proteins......Page 224
Human Rrp45/Rrp41......Page 225
Human Rrp46......Page 226
Reconstitution and Purification of Human and Yeast Exosomes......Page 227
The yeast ten-subunit exosome......Page 228
Further purification of yeast exosomes......Page 229
The human nine-subunit exosome......Page 231
Exoribonuclease Assays......Page 232
Comparative Exoribonuclease Assays with Different RNA Substrates......Page 233
Acknowledgments......Page 234
References......Page 235
Biochemical Studies of the Mammalian Exosome with Intact Cells......Page 237
Introduction......Page 238
Identifying Protein-Protein Interactions by the Mammalian Two-Hybrid System......Page 239
Protocol......Page 240
Comments......Page 242
Characterization of Different Exosome Subsets by Glycerol Sedimentation......Page 244
Preparation of 5 to 40% glycerol gradients......Page 245
Comments......Page 246
Protocol......Page 248
Comments......Page 249
References......Page 250
Determining In Vivo Activity of the Yeast Cytoplasmic Exosome......Page 253
Introduction......Page 254
Core exosome mutants......Page 255
Nuclear exosome cofactors mutants......Page 257
Protocol......Page 258
The use of synthetic lethality to analyze the degradation of normal transcripts by the cytoplasmic exosome......Page 260
Protocol......Page 261
The use of the killer assay to analyze the activity of the cytoplasmic exosome......Page 262
Protocol......Page 263
References......Page 264
Approaches for Studying PMR1 Endonuclease-mediated mRNA Decay......Page 266
Introduction......Page 267
Overview......Page 269
Generation of single-stranded probes by asymmetric PCR......Page 270
S1 nuclease protection......Page 271
Protocol......Page 272
Primer ligation......Page 274
RT-PCR......Page 275
Analysis of PMR1-Containing Complexes......Page 276
Preparation of postmitochondrial extracts......Page 277
Linear sucrose density gradients......Page 278
Sucrose step gradients......Page 280
Glycerol gradient analysis of PMR1 containing complexes......Page 281
Affinity Recovery of PMR1-Containing Complexes......Page 282
Sample application, washing, and elution with TEV protease......Page 283
Immunoprecipitation of PMR1 with immobilized anti-myc antibody......Page 284
Protocol......Page 285
In vitro analysis of PMR1 activity......Page 286
References......Page 287
Methods to Determine mRNA Half-Life in Saccharomyces cerevisiae......Page 289
Introduction......Page 290
The GAL1 UAS......Page 291
Transcriptional shut-off......Page 292
SGS Medium (1 L)......Page 293
Transcriptional pulse-chase......Page 294
SR Medium (1 L)......Page 296
The TET-off system......Page 297
Measuring mRNA Decay by Use of Thermally Labile Alleles of RNA Polymerase II......Page 298
RNA Extractions......Page 299
RNase H cleavages of 3\' UTRs......Page 300
2x RNase H buffer......Page 301
Determination of mRNA Half-Lives......Page 302
References......Page 304
mRNA Decay Analysis in Drosophila melanogaster: Drug-Induced Changes in Glutathione S-Transferase D21 mRNA Stability......Page 307
Introduction......Page 308
Transgenic constructs and nomenclature......Page 309
Microinjection and establishment of transgenic lines......Page 311
Pentobarbital and heat shock treatments......Page 312
RNA isolation and RNase Protection Assays......Page 313
Determining 5\'- and/or 3\'-ends and decay intermediates of gstD21 mRNAs......Page 315
Determination of gstD21 mRNA half-lives......Page 316
Concluding Remarks......Page 317
References......Page 318
Measuring mRNA Stability During Early Drosophila Embryogenesis......Page 320
Maternal mRNAs and Early Drosophila Development......Page 321
Dual degradation activities in the early embryo......Page 322
Studying maternal mRNA decay in unfertilized eggs versus fertilized embryos......Page 323
Prerequisites for triggering maternal mRNA destabilization......Page 328
Description and comparison of RNA methods......Page 329
Analysis of deadenylation......Page 335
Sample collection......Page 336
Total RNA extraction with TRIzol......Page 337
Single-embryo dot blot analysis......Page 338
RNA isolation......Page 339
\"Direct\" versus \"indirect\" labeling methods......Page 340
Data normalization......Page 341
Postscanning normalization......Page 342
Establishing transcripts present in the reference sample......Page 343
Identifying transcripts undergoing degradation......Page 344
Indirect reverse transcription master mix......Page 346
Procedure......Page 348
Scanning and quantification and analysis of microarray data......Page 349
Acknowledgments......Page 352
References......Page 353
Messenger RNA Half-Life Measurements in Mammalian Cells......Page 356
Introduction......Page 357
General Considerations of mRNA Half-Life Measurements......Page 358
Determining mRNA Decay Constant......Page 359
General inhibition of transcription......Page 360
Use of inducible promoters to specifically promote transient transcription......Page 361
The c-fos serum-inducible promoter system......Page 362
Materials......Page 363
Procedures......Page 364
Procedure......Page 365
Materials......Page 366
Procedure......Page 367
Protocol II: Transient transfection and serum induction......Page 369
The Tet-off regulatory promoter system......Page 370
Establishment of mammalian stable cell lines expressing the tTA......Page 371
Transcriptional pulse strategy by modulating the amount of tetracycline in culture medium......Page 372
Transfection procedure......Page 373
Directly measuring mRNA half-life with the Tet-off promoter system without transcriptional pulsing......Page 374
Concluding Remarks......Page 375
References......Page 376
Introduction......Page 379
In situ tagging......Page 381
Troubleshooting......Page 382
Overview......Page 383
The cells......Page 384
Time course......Page 385
Overview......Page 386
Procedure......Page 387
Procedure......Page 388
Overview......Page 389
Labeling procedure......Page 390
Material required......Page 391
Sample prehybridization......Page 392
Materials required......Page 393
Hybridization (first day)......Page 394
References......Page 395
Cell Type-Specific Analysis of mRNA Synthesis and Decay In Vivo with Uracil Phosphoribosyltransferase and 4-thiouracil......Page 398
Introduction......Page 399
Targeted expression of TgUPRT......Page 401
General RNA tagging with 4-thiouridine......Page 403
Purification and analysis of 4TU-tagged RNA......Page 404
RNA preparation......Page 405
Purification of 4TU-tagged RNA......Page 406
4TU pulse......Page 407
Tips and controls for the 4TU pulse and uracil chase......Page 408
Total RNA extraction......Page 409
Tips and controls for RNA preparation......Page 410
Precipitation of biotinylated RNA......Page 411
RNA-blot for detection of 4TU-tagged RNA......Page 412
Run biotinylated RNA on agarose gel......Page 413
Quantification of amounts of 4TU-tagged RNA......Page 414
Tips and controls for RNA blots......Page 415
Purify biotinylated 4TU-tagged RNA with streptavidin-magnetic beads......Page 416
Precipitate the RNA......Page 417
Tips and controls......Page 418
Microarray analysis: Design and normalization considerations......Page 419
Experimental design for the analysis of mRNA synthesis versus abundance......Page 420
Direct comparison of samples versus hybridization against a common reference......Page 421
Normalization with RNA blot data......Page 422
Analysis of pulse-chase microarray data......Page 423
References......Page 424
Analysis of Cytoplasmic mRNA Decay in Saccharomyces cerevisiae......Page 426
Measuring mRNA Half-Life......Page 427
Use of the galactose promoter......Page 430
Use of the tetracycline-regulatable promoter system......Page 431
Determination of mRNA Decay Pathways......Page 432
Trapping mRNA decay intermediates......Page 433
Determining the directionality of a decay pathway......Page 434
Trapped decay intermediates as a simple assay for 3\' to 5\'-mRNA decay......Page 435
Determining precursor-product relationships in a transcriptional pulse-chase......Page 436
Strains defective in 5\' to 3\'-decay......Page 439
Strains specifically affecting specialized mRNA decay pathways......Page 441
References......Page 442
Exosome: At the Nexus of the Cellular RNA Transactions......Page 445
Unique Features of the Plant Exosome......Page 448
Resources for the Mutational Analyses of the Plant Exosome......Page 450
Transcriptome-wide Mapping of Targets of the Plant Exosome Complex......Page 452
References......Page 456
Sensitive Detection of mRNA Decay Products by Use of Reverse-Ligation-Mediated PCR (RL-PCR)......Page 460
Introduction......Page 461
Footprinting of RNA-Protein Interaction......Page 465
Procedure......Page 466
RL-PCR with Ligation of an RNA Linker......Page 467
Procedure......Page 469
Preliminary treatments of the 5\'-ends of the total cellular RNA......Page 470
RL-PCR......Page 471
Primer labeling for the analysis of 10 samples (scale up if necessary)......Page 472
Circularization RL-PCR to Analyze mRNA Decay Involving Modification of the 5\'- and 3\'-Ends......Page 473
Procedure......Page 475
Circularization RL-PCR......Page 477
cDNA synthesis......Page 478
Primer labeling for the analysis of 20 samples......Page 479
References......Page 480
Tethering Assays to Investigate Nonsense-Mediated mRNA Decay Activating Proteins......Page 482
Introduction......Page 483
Effector plasmid......Page 485
Reporter plasmid......Page 487
Insertion of boxB or MS2 sites into the reporter mRNA......Page 488
Transfection control plasmid......Page 489
Buffers and solutions......Page 490
Preparation of total-cytoplasmic or total-cell RNA......Page 491
Capillary transfer......Page 492
Alternative detection methods......Page 493
Control experiments......Page 494
References......Page 495
Assays for Determining Poly(A) Tail Length and the Polarity of mRNA Decay in Mammalian Cells......Page 498
RNA preparation......Page 499
cDNA synthesis......Page 500
PCR......Page 501
Materials: LM-PAT assay......Page 503
Reverse transcription......Page 504
Materials: RNase H assay......Page 505
Visualization of RNA products......Page 506
Introduction: Invader RNA Assay......Page 507
In vitro synthesized transcript of the gene of interest......Page 509
Primary reaction oligonucleotide design, synthesis, and preparation......Page 510
Secondary reaction oligonucleotide design, synthesis, and preparation......Page 512
Experimental design considerations......Page 513
RNA isolation......Page 514
Preparation of the standard curve......Page 515
Preparation of reaction mixes......Page 516
Data analysis, standard curves......Page 517
Data analysis, mRNA decay......Page 518
References......Page 519
Analyzing P-bodies in Saccharomyces cerevisiae......Page 520
Markers of P-bodies......Page 521
Examination of P-bodies in midlog growth......Page 525
Examination of P-bodies under glucose deprivation or osmotic stress......Page 526
Monitoring Messenger RNA in P-Bodies......Page 527
Conditions to observe increases or decreases in P-bodies......Page 528
Interpreting alterations in P-body size and number......Page 530
Quantification of P-Body Size and Number......Page 531
References......Page 532
Real-Time and Quantitative Imaging of Mammalian Stress Granules and Processing Bodies......Page 534
Introduction......Page 535
Choice of fluorescent tag......Page 537
Reporter construct design......Page 539
Selection Criteria......Page 540
Transfection......Page 541
Drug selection......Page 542
Picking clones......Page 543
Subcloning procedure......Page 544
Clone expansion procedure......Page 545
Properties of Representative Stable Lines......Page 546
Advantages......Page 554
Advantages......Page 555
Microscope Hardware: Widefield vs Confocal......Page 556
Advantages......Page 558
Disadvantages......Page 559
Live cell imaging for tracking processing bodies......Page 560
Live cell imaging for single-image quantification......Page 562
Acquiring FRAP/FLIP images......Page 563
References......Page 564
Cell Biology of mRNA Decay......Page 566
Introduction......Page 567
FISH Probe Design......Page 569
Image Acquisition......Page 570
FISH Protocol......Page 571
Wash......Page 572
Colabeling Protein with IF and RNA with FISH......Page 573
FISH hybridization......Page 574
Following mRNA in Living Cells......Page 575
Live Single-Molecule Detection......Page 576
Single mRNA Data Analysis; What You Can Observe......Page 577
How Do You Know That You See Single Molecules?......Page 579
The Secret to Getting Good Data: More Photons, Less Noise......Page 581
Setting Up a Microscope for Single Molecule Detection......Page 582
Appendix......Page 584
Experimental Controls......Page 587
References......Page 588
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