RNA Turnover in Eukaryotes: Nucleases, Pathways and Analysis of mRNA Decay (Volume 448) (Methods in Enzymology, Volume 448)

Specific complexes of protein and RNA carry out many essential biological functions, including RNA processing, RNA turnover, RNA folding, as well as the translation of genetic information from mRNA into protein sequences. Messenger RNA (mRNA) decay is now emerging as an important control point and a major contributor to gene expression. Continuing identification of the protein factors and cofactors, and mRNA instability elements, responsible for mRNA decay allow researchers to build a comprehensive picture of the highly orchestrated processes involved in mRNA decay and its regulation. The control of biological processes, such as cellular growth and differentiation, is dependent on how the genetic material within a cell is expressed. The cellular physiology of mRNA-including mRNA processing, transport, localization, and turnover-is central to the process of gene expression. Covers the nonsense-mediated mRNA decay (NMD) or mRNA surveillance pathway. Expert researchers introduce the most advanced technologies and techniques to identify mRNA processing, transport, localization and turnover which are central to the process of gene expression. Offers step-by-step lab instructions including necessary equipment and reagents 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 B......Page 592 C......Page 594 E......Page 595 G......Page 596 H......Page 597 J......Page 598 K......Page 599 L......Page 600 M......Page 601 O......Page 602 R......Page 603 S......Page 604 U......Page 606 W......Page 607 Z......Page 608 D......Page 610 I......Page 612 M......Page 613 P......Page 615 R......Page 617 T......Page 618 X......Page 619 Z......Page 620 Analysis of mRNA decapping / S.W. Liu ... [et al.] A kinetic assay to monitor RNA decapping under single- turnover conditions / B.N. Jones ... [et al.] Purification and analysis of the decapping activator Lsm1p-7p-Pat1p complex from yeast / S. Tharun Reconstitution of recombinant human LSm complexes for biochemical, biophysical, and cell biological studies / B.L. Zaric and C. Kambach Regulated deadenylation in vitro / A.C. Goldstrohm, B.A. Hook and M. Wickens Cell-free deadenylation assays with Drosophila embryo extracts / M. Jeske and E. Wahle Measuring CPEB-mediated cytoplasmic polyadenylation-deadenylation in Xenopus laevis oocytes and egg extracts / J.H. Kim and J.D. Richter The preparation and applications of cytoplasmic extracts from mammalian cells for studying aspects of mRNA decay / K.J. Sokoloski, J. Wilusz and C.J. Wilusz In vitro assays of 5' to 3'-exoribonuclease activity / O. Pellegrini ... [et al.] ^ Reconstitution of RNA exosomes from human and Saccharomyces cerevisiae cloning, expression, purification, and activity assays / J.C. Greimann and C.D. Lima Biochemical studies of the mammalian exosome with intact cells / G. Schilders and G.J. Pruijn Determining in vivo activity of the yeast cytoplasmic exosome / D. Schaeffer ... [et al.] Approaches for studying PMR1 endonuclease-mediated mRNA decay / Y. Otsuka and D.R. Schoenberg Methods to determine mRNA half-life in Saccharomyces cerevisiae / J. Coller mRNA Decay analysis in Drosophila melanogaster drug-induced changes in glutathione S-transferase D21 mRNA stability / B. Akgul and C.P. Tu Measuring mRNA stability during early Drosophila embryogenesis / J.L. Semotok ... [et al.] Messenger RNA half-life measurements in mammalian cells / C.Y. Chen, N. Ezzeddine and A.B. Shyu Trypanosomes as a model to investigate mRNA decay pathways / S. Archer ... [et al.] ^ ^^ Cell type-specific analysis of mRNA synthesis and decay in vivo with uracil phosphoribosyltransferase and 4-thiouracil / M.D. Cleary Analysis of cytoplasmic mRNA decay in Saccharomyces cerevisiae / D.O. Passos and R. Parker Transcriptome targets of the exosome complex in plants / D. Belostotsky Sensitive detection of mRNA decay products by use of reverse-ligation-mediated PCR (RL-PCR) / T. Grange Tethering assays to investigate nonsense-mediated mRNA decay activating proteins / N.H. Gehring, M.W. Hentze and A.E. Kulozik Assays for determining poly(A) tail length and the polarity of mRNA decay in mammalian cells / E.L. Murray and D.R. Schoenberg Analyzing P-bodies in Saccharomyces cerevisiae / T. Nissan and R. Parker Real-time and quantitative imaging of mammalian stress granules and processing bodies / N. Kedersha ... [et al.] Cell biology of mRNA decay / D. Grunwald, R.H. Singer and K. Czaplinski. ^^ Specific complexes of protein and RNA carry out many essential biological functions, including RNA processing, RNA turnover, RNA folding, as well as the translation of genetic information from mRNA into protein sequences. Messenger RNA (mRNA) decay is now emerging as an important control point and a major contributor to gene expression. Continuing identification of the protein factors and cofactors, and mRNA instability elements responsible for mRNA decay allow researchers to build a comprehensive picture of the highly orchestrated processes involved in mRNA decay and its regulation.

Covers the nonsense-mediated mRNA decay (NMD) or mRNA surveillance pathway
Expert researchers introduce the most advanced technologies and techniques to identify mRNA processing, transport, localization and turnover, which are central to the process of gene expression
Offers step-by-step lab instructions, including necessary equipment and reagents