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

Cover Page......Page 1
Series Editors......Page 2
Methods in Enzymology......Page 0
Copyright Page......Page 3
Contributors......Page 4
Preface......Page 10
Methods in Enzymology......Page 12
Analysis of mRNA Decapping......Page 38
Introduction......Page 39
Measuring Decapping Activities of Recombinant and Endogenous Dcp2......Page 41
In vitro Transcription......Page 42
Gel purification of the cap-labeled RNA......Page 43
Measuring Dcp2 decapping activity......Page 44
Preparation of recombinant flag-tagged hDcp2 expressed in human cells......Page 45
Preparation of endogenous hDcp2 (P50 cytoplasmic fraction) for decapping assays......Page 46
In vitro Dcp2 decapping assay......Page 47
Presence of a copurifying bacterial pyrophosphatase activity......Page 49
Measuring DcpS Activity......Page 50
Generation of total-cell extract to detect endogenous DcpS activity......Page 51
Migration of Cap Analogs with Various Thin-Layer Chromatography Running Buffers......Page 52
Preparation of m7GTP......Page 53
References......Page 54
A Kinetic Assay to Monitor RNA Decapping Under Single-Turnover Conditions......Page 57
Introduction......Page 58
Kinetic Equations......Page 59
Cap-labeled RNA preparation......Page 62
Substrate characterization......Page 63
Overview......Page 64
TLC analysis......Page 66
Curve fitting......Page 67
Metal activation......Page 68
Summary......Page 70
Buffers......Page 71
References......Page 72
Purification and Analysis of the Decapping Activator Lsm1p-7p-Pat1p Complex from Yeast......Page 75
Introduction......Page 76
Purification of the Lsm1p-7p-Pat1p Complex......Page 77
Buffers (prepared with the stock solutions listed above)......Page 79
Preclearing of lysate......Page 80
Ni-NTA matrix binding and elution......Page 81
Analysis of RNA Binding by the Lsm1p-7p-Pat1p Complex......Page 84
References......Page 87
Reconstitution of Recombinant Human LSm Complexes for Biochemical, Biophysical, and Cell Biological Studies......Page 90
Introduction......Page 91
Cloning......Page 94
Protein Expression and Purification......Page 98
LSm Complex Reconstitution......Page 101
LSm Complex Functional Assays......Page 103
References......Page 105
Regulated Deadenylation in Vitro......Page 108
Introduction......Page 109
Advantages......Page 111
A brief history......Page 112
Optimizing deadenylation conditions......Page 113
RNA substrates......Page 115
5'-End labeling......Page 116
Protocol: 5'-labeling of synthetic substrate RNA......Page 117
Purified components......Page 118
Harvest cells......Page 119
Exchange buffer......Page 121
Control reactions......Page 122
Analyze products on a denaturing polyacrylamide gel......Page 124
Analyze purified regulator......Page 125
Test activity of purified regulator......Page 126
Interpret data......Page 127
Assay-regulated deadenylation in vitro......Page 128
Interpret data......Page 130
Regulator concentration......Page 131
References......Page 132
Introduction......Page 138
Preparation of Drosophila Embryo Extracts......Page 141
Preparation of Substrate RNA......Page 143
Deadenylation Assay with Drosophila Embryo Extracts......Page 144
Characterization of Sequence-Dependent Deadenylation in Drosophila Embryo Extracts......Page 146
References......Page 147
Measuring CPEB-Mediated Cytoplasmic Polyadenylation-Deadenylation in Xenopus laevis Oocytes and Egg Extracts......Page 150
Introduction......Page 151
Principle......Page 152
Oocyte collection, injection, incubation, and retrieval of radiolabeled RNA probes......Page 153
Methods......Page 154
Notes......Page 156
Methods......Page 157
Principle......Page 159
Notes......Page 160
Methods......Page 161
Notes......Page 162
Methods......Page 163
Depletion of protein with specific antibody and polyadenylation assay......Page 164
Egg extract preparation......Page 165
Depletion of protein with specific antibody and polyadenylation assay......Page 166
Depletion of protein with specific interacting protein and polyadenylation assay......Page 167
References......Page 168
The Preparation and Applications of Cytoplasmic Extracts from Mammalian Cells for Studying Aspects of mRNA Decay......Page 170
Introduction......Page 171
Preparation of HeLa-Cell Cytoplasmic Extracts......Page 173
Preparation of HeLa-Cell S100 cytoplasmic extracts......Page 174
Standardization of cytoplasmic extract activity......Page 176
Protocol......Page 178
Transcription of polyadenylated and nonadenylated RNA substrates......Page 179
Production of cap-labeled RNA substrate......Page 182
Protocol......Page 183
Determining exonuclease activity......Page 184
Protocol......Page 186
Protocol......Page 187
Analysis of trans-acting factors with ultraviolet crosslinking......Page 188
Protocol......Page 189
Immunoprecipitation protocol......Page 191
Concluding Remarks......Page 192
References......Page 193
In Vitro Assays of 5' to 3'-Exoribonuclease Activity......Page 195
Introduction......Page 196
Purification of Xrn1......Page 198
In Vitro RNA Substrate Synthesis......Page 199
TCA-based exoribonuclease assays......Page 201
Gel-based exoribonuclease assays......Page 202
Degradation of doubly labeled RNA by Xrn1......Page 204
Degradation of 5'- and 3'-labeled synthetic RNAs by Xrn1......Page 207
Conclusions and Prospects......Page 208
References......Page 209
Reconstitution of RNA Exosomes from Human and Saccharomyces cerevisiae: Cloning, Expression, Purification, and Activity Assays......Page 212
Introduction......Page 213
Cloning Strategies for Recombinant Protein Expression......Page 217
Yeast RRP41/RRP45 cDNA......Page 218
Yeast RRP6 cDNA fused to SMT3 cDNA......Page 220
Human RRP4, RRP40, and CSL4 cDNA......Page 221
Expression and Purification of Yeast Exosome Proteins......Page 222
Yeast Mtr3/Rrp42......Page 223
Yeast Rrp44......Page 224
Expression and Purification of Human Exosome Proteins......Page 225
Human Rrp45/Rrp41......Page 226
Human Rrp46......Page 227
Reconstitution and Purification of Human and Yeast Exosomes......Page 228
The yeast ten-subunit exosome......Page 229
Further purification of yeast exosomes......Page 230
The human nine-subunit exosome......Page 232
Exoribonuclease Assays......Page 233
Comparative Exoribonuclease Assays with Different RNA Substrates......Page 234
Acknowledgments......Page 235
References......Page 236
Biochemical Studies of the Mammalian Exosome with Intact Cells......Page 238
Introduction......Page 239
Identifying Protein-Protein Interactions by the Mammalian Two-Hybrid System......Page 240
Protocol......Page 241
Comments......Page 243
Characterization of Different Exosome Subsets by Glycerol Sedimentation......Page 245
Preparation of 5 to 40% glycerol gradients......Page 246
Comments......Page 247
Protocol......Page 249
Comments......Page 250
References......Page 251
Determining In Vivo Activity of the Yeast Cytoplasmic Exosome......Page 254
Introduction......Page 255
Core exosome mutants......Page 256
Nuclear exosome cofactors mutants......Page 258
Protocol......Page 259
The use of synthetic lethality to analyze the degradation of normal transcripts by the cytoplasmic exosome......Page 261
Protocol......Page 262
The use of the killer assay to analyze the activity of the cytoplasmic exosome......Page 263
Protocol......Page 264
References......Page 265
Approaches for Studying PMR1 Endonuclease-mediated mRNA Decay......Page 267
Introduction......Page 268
Overview......Page 270
Generation of single-stranded probes by asymmetric PCR......Page 271
S1 nuclease protection......Page 272
Protocol......Page 273
Primer ligation......Page 275
RT-PCR......Page 276
Analysis of PMR1-Containing Complexes......Page 277
Preparation of postmitochondrial extracts......Page 278
Linear sucrose density gradients......Page 279
Sucrose step gradients......Page 281
Glycerol gradient analysis of PMR1 containing complexes......Page 282
Affinity Recovery of PMR1-Containing Complexes......Page 283
Sample application, washing, and elution with TEV protease......Page 284
Immunoprecipitation of PMR1 with immobilized anti-myc antibody......Page 285
Protocol......Page 286
In vitro analysis of PMR1 activity......Page 287
References......Page 288
Methods to Determine mRNA Half-Life in Saccharomyces cerevisiae......Page 290
Introduction......Page 291
The GAL1 UAS......Page 292
Transcriptional shut-off......Page 293
SGS Medium (1 L)......Page 294
Transcriptional pulse-chase......Page 295
SR Medium (1 L)......Page 297
The TET-off system......Page 298
Measuring mRNA Decay by Use of Thermally Labile Alleles of RNA Polymerase II......Page 299
RNA Extractions......Page 300
RNase H cleavages of 3' UTRs......Page 301
2x RNase H buffer......Page 302
Determination of mRNA Half-Lives......Page 303
References......Page 305
mRNA Decay Analysis in Drosophila melanogaster: Drug-Induced Changes in Glutathione S-Transferase D21 mRNA Stability......Page 308
Introduction......Page 309
Transgenic constructs and nomenclature......Page 310
Microinjection and establishment of transgenic lines......Page 312
Pentobarbital and heat shock treatments......Page 313
RNA isolation and RNase Protection Assays......Page 314
Determining 5'- and/or 3'-ends and decay intermediates of gstD21 mRNAs......Page 316
Determination of gstD21 mRNA half-lives......Page 317
Concluding Remarks......Page 318
References......Page 319
Measuring mRNA Stability During Early Drosophila Embryogenesis......Page 321
Maternal mRNAs and Early Drosophila Development......Page 322
Dual degradation activities in the early embryo......Page 323
Studying maternal mRNA decay in unfertilized eggs versus fertilized embryos......Page 324
Prerequisites for triggering maternal mRNA destabilization......Page 329
Description and comparison of RNA methods......Page 330
Analysis of deadenylation......Page 336
Sample collection......Page 337
Total RNA extraction with TRIzol......Page 338
Single-embryo dot blot analysis......Page 339
RNA isolation......Page 340
"Direct" versus "indirect" labeling methods......Page 341
Data normalization......Page 342
Postscanning normalization......Page 343
Establishing transcripts present in the reference sample......Page 344
Identifying transcripts undergoing degradation......Page 345
Indirect reverse transcription master mix......Page 347
Procedure......Page 349
Scanning and quantification and analysis of microarray data......Page 350
Acknowledgments......Page 353
References......Page 354
Messenger RNA Half-Life Measurements in Mammalian Cells......Page 357
Introduction......Page 358
General Considerations of mRNA Half-Life Measurements......Page 359
Determining mRNA Decay Constant......Page 360
General inhibition of transcription......Page 361
Use of inducible promoters to specifically promote transient transcription......Page 362
The c-fos serum-inducible promoter system......Page 363
Materials......Page 364
Procedures......Page 365
Procedure......Page 366
Materials......Page 367
Procedure......Page 368
Protocol II: Transient transfection and serum induction......Page 370
The Tet-off regulatory promoter system......Page 371
Establishment of mammalian stable cell lines expressing the tTA......Page 372
Transcriptional pulse strategy by modulating the amount of tetracycline in culture medium......Page 373
Transfection procedure......Page 374
Directly measuring mRNA half-life with the Tet-off promoter system without transcriptional pulsing......Page 375
Concluding Remarks......Page 376
References......Page 377
Introduction......Page 380
In situ tagging......Page 382
Troubleshooting......Page 383
Overview......Page 384
The cells......Page 385
Time course......Page 386
Overview......Page 387
Procedure......Page 388
Procedure......Page 389
Overview......Page 390
Labeling procedure......Page 391
Material required......Page 392
Sample prehybridization......Page 393
Materials required......Page 394
Hybridization (first day)......Page 395
References......Page 396
Cell Type-Specific Analysis of mRNA Synthesis and Decay In Vivo with Uracil Phosphoribosyltransferase and 4-thiouracil......Page 399
Introduction......Page 400
Targeted expression of TgUPRT......Page 402
General RNA tagging with 4-thiouridine......Page 404
Purification and analysis of 4TU-tagged RNA......Page 405
RNA preparation......Page 406
Purification of 4TU-tagged RNA......Page 407
4TU pulse......Page 408
Tips and controls for the 4TU pulse and uracil chase......Page 409
Total RNA extraction......Page 410
Tips and controls for RNA preparation......Page 411
Precipitation of biotinylated RNA......Page 412
RNA-blot for detection of 4TU-tagged RNA......Page 413
Run biotinylated RNA on agarose gel......Page 414
Quantification of amounts of 4TU-tagged RNA......Page 415
Tips and controls for RNA blots......Page 416
Purify biotinylated 4TU-tagged RNA with streptavidin-magnetic beads......Page 417
Precipitate the RNA......Page 418
Tips and controls......Page 419
Microarray analysis: Design and normalization considerations......Page 420
Experimental design for the analysis of mRNA synthesis versus abundance......Page 421
Direct comparison of samples versus hybridization against a common reference......Page 422
Normalization with RNA blot data......Page 423
Analysis of pulse-chase microarray data......Page 424
References......Page 425
Analysis of Cytoplasmic mRNA Decay in Saccharomyces cerevisiae......Page 427
Measuring mRNA Half-Life......Page 428
Use of the galactose promoter......Page 431
Use of the tetracycline-regulatable promoter system......Page 432
Determination of mRNA Decay Pathways......Page 433
Trapping mRNA decay intermediates......Page 434
Determining the directionality of a decay pathway......Page 435
Trapped decay intermediates as a simple assay for 3' to 5'-mRNA decay......Page 436
Determining precursor-product relationships in a transcriptional pulse-chase......Page 437
Strains defective in 5' to 3'-decay......Page 440
Strains specifically affecting specialized mRNA decay pathways......Page 442
References......Page 443
Exosome: At the Nexus of the Cellular RNA Transactions......Page 446
Unique Features of the Plant Exosome......Page 449
Resources for the Mutational Analyses of the Plant Exosome......Page 451
Transcriptome-wide Mapping of Targets of the Plant Exosome Complex......Page 453
References......Page 457
Sensitive Detection of mRNA Decay Products by Use of Reverse-Ligation-Mediated PCR (RL-PCR)......Page 461
Introduction......Page 462
Footprinting of RNA-Protein Interaction......Page 466
Procedure......Page 467
RL-PCR with Ligation of an RNA Linker......Page 468
Procedure......Page 470
Preliminary treatments of the 5'-ends of the total cellular RNA......Page 471
RL-PCR......Page 472
Primer labeling for the analysis of 10 samples (scale up if necessary)......Page 473
Circularization RL-PCR to Analyze mRNA Decay Involving Modification of the 5'- and 3'-Ends......Page 474
Procedure......Page 476
Circularization RL-PCR......Page 478
cDNA synthesis......Page 479
Primer labeling for the analysis of 20 samples......Page 480
References......Page 481
Tethering Assays to Investigate Nonsense-Mediated mRNA Decay Activating Proteins......Page 483
Introduction......Page 484
Effector plasmid......Page 486
Reporter plasmid......Page 488
Insertion of boxB or MS2 sites into the reporter mRNA......Page 489
Transfection control plasmid......Page 490
Buffers and solutions......Page 491
Preparation of total-cytoplasmic or total-cell RNA......Page 492
Capillary transfer......Page 493
Alternative detection methods......Page 494
Control experiments......Page 495
References......Page 496
Assays for Determining Poly(A) Tail Length and the Polarity of mRNA Decay in Mammalian Cells......Page 499
RNA preparation......Page 500
cDNA synthesis......Page 501
PCR......Page 502
Materials: LM-PAT assay......Page 504
Reverse transcription......Page 505
Materials: RNase H assay......Page 506
Visualization of RNA products......Page 507
Introduction: Invader RNA Assay......Page 508
In vitro synthesized transcript of the gene of interest......Page 510
Primary reaction oligonucleotide design, synthesis, and preparation......Page 511
Secondary reaction oligonucleotide design, synthesis, and preparation......Page 513
Experimental design considerations......Page 514
RNA isolation......Page 515
Preparation of the standard curve......Page 516
Preparation of reaction mixes......Page 517
Data analysis, standard curves......Page 518
Data analysis, mRNA decay......Page 519
References......Page 520
Analyzing P-bodies in Saccharomyces cerevisiae......Page 521
Markers of P-bodies......Page 522
Examination of P-bodies in midlog growth......Page 526
Examination of P-bodies under glucose deprivation or osmotic stress......Page 527
Monitoring Messenger RNA in P-Bodies......Page 528
Conditions to observe increases or decreases in P-bodies......Page 529
Interpreting alterations in P-body size and number......Page 531
Quantification of P-Body Size and Number......Page 532
References......Page 533
Real-Time and Quantitative Imaging of Mammalian Stress Granules and Processing Bodies......Page 535
Introduction......Page 536
Choice of fluorescent tag......Page 538
Reporter construct design......Page 540
Selection Criteria......Page 541
Transfection......Page 542
Drug selection......Page 543
Picking clones......Page 544
Subcloning procedure......Page 545
Clone expansion procedure......Page 546
Properties of Representative Stable Lines......Page 547
Advantages......Page 555
Advantages......Page 556
Microscope Hardware: Widefield vs Confocal......Page 557
Advantages......Page 559
Disadvantages......Page 560
Live cell imaging for tracking processing bodies......Page 561
Live cell imaging for single-image quantification......Page 563
Acquiring FRAP/FLIP images......Page 564
References......Page 565
Cell Biology of mRNA Decay......Page 567
Introduction......Page 568
FISH Probe Design......Page 570
Image Acquisition......Page 571
FISH Protocol......Page 572
Wash......Page 573
Colabeling Protein with IF and RNA with FISH......Page 574
FISH hybridization......Page 575
Following mRNA in Living Cells......Page 576
Live Single-Molecule Detection......Page 577
Single mRNA Data Analysis; What You Can Observe......Page 578
How Do You Know That You See Single Molecules?......Page 580
The Secret to Getting Good Data: More Photons, Less Noise......Page 582
Setting Up a Microscope for Single Molecule Detection......Page 583
Appendix......Page 585
Experimental Controls......Page 588
References......Page 589
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