RNA Turnover in Bacteria, Archaea and Organelles

Cover Page......Page 1
Series Editors......Page 2
Methods in Enzymology......Page 0
Copyright Page......Page 3
Contributors to Volume 447......Page 4
Preface......Page 12
Volumes in Series......Page 14
Analysis of RNA Decay, Processing, and Polyadenylation in Escherichia coli and Other Prokaryotes......Page 40
Introduction......Page 41
General Considerations When Working with RNA......Page 42
Kits versus detergent methods for RNA isolation......Page 43
Reagents and buffers......Page 44
Procedure......Page 45
RNA extraction......Page 46
Procedure......Page 47
Removal of DNA Contamination......Page 48
RNA Integrity Assessment......Page 49
Isolation of Polyadenylated RNA......Page 50
Northern Analysis......Page 51
Separation of RNA in agarose gels......Page 52
Choice of probes......Page 53
Probe labeling......Page 54
Procedure......Page 55
Stripping of hybridized probes......Page 56
Procedure......Page 57
Methods......Page 59
Reverse Transcription PCR (RT-PCR) to Analyze 3prime -ends of Specific Transcripts......Page 60
Cloning and sequencing of PCR products......Page 62
RNA self-ligation for determining 3prime - and 5prime -ends......Page 64
References......Page 65
Analyzing the Decay of Stable RNAs in E. coli......Page 67
Introduction......Page 68
Preparation of Stable RNA Substrates for In Vitro Degradation Assays......Page 71
Detection of Degradation Products In Vitro......Page 73
Examination of Stable RNA Decay In Vivo......Page 74
Determination of the 3prime- and 5prime-Termini of Intermediates during the Processing and Degradation of Stable RNAs......Page 75
Concluding Remarks......Page 79
References......Page 80
Genomic Analysis of mRNA Decay in E. coli with DNA Microarrays......Page 82
Introduction......Page 83
Experimental design......Page 84
Control spots for normalization......Page 85
Cell harvest......Page 86
RNA extraction and quantification......Page 87
Prepare first strand amino-allyl cDNA(aa-cDNA) with amino-allyl-dUTP......Page 88
Purify Alexa Dye-aa-cDNA with Qiagen QIAquick PCR purification kit......Page 89
PCR product verification......Page 90
Slide postprocessing......Page 91
Hybridization......Page 92
Microarray scanning......Page 93
Creating a data set for half-life calculation......Page 94
References......Page 97
Co-immunopurification of Multiprotein Complexes Containing RNA-Degrading Enzymes......Page 100
Introduction......Page 101
General Considerations......Page 103
Materials......Page 106
Cross-linking IgG to protein A Sepharose beads......Page 107
Co-immunopurification with antibodies coupled to protein A beads......Page 108
Small-scale affinity purification of polyclonal antibodies......Page 110
Perspective......Page 111
Characterization of the RNase E of psychrotolerant bacteria......Page 112
Acknowledgments......Page 114
References......Page 115
PABLO Analysis of RNA: 5prime-Phosphorylation State and 5prime-End Mapping......Page 118
Concept......Page 119
Detection Method......Page 121
Determining the 5prime-Phosphorylation State of Long Transcripts......Page 122
Ascertaining Whether the Decay of a Transcript Begins with Pyrophosphate Removal......Page 124
PABLO ligation and analysis......Page 126
Ligation......Page 127
In vitro synthesis of internally radiolabeled RNA bearing a 5prime -monophosphate......Page 128
Probing the phosphorylation state of RNA with a 5prime-monophosphate-dependent exonuclease......Page 129
The Use of PABLO to Map the 5prime-End of RNA......Page 130
References......Page 132
A Proteomic Approach to the Analysis of RNA Degradosome Composition in Escherichia coli......Page 134
The RNA degradosome......Page 135
Proteomic approaches to the analysis of complex protein mixtures......Page 136
RNA degradosome purification......Page 140
Enzymatic digestion......Page 141
MudPIT analysis......Page 142
Differential analysis......Page 145
Validation......Page 147
Perspective and Conclusion......Page 148
References......Page 150
New Approaches to Understanding Double-Stranded RNA Processing by Ribonuclease III: Purification and Assays of Homodimeric and Heterodimeric Forms of RNase III from Bacterial Extremophiles and Mesophiles......Page 153
Introduction......Page 154
Heterologous Expression, Affinity Purification, and Assays of Aquifex aeolicus and Thermotoga maritima RNases III......Page 156
Substrate cleavage assay......Page 158
Production and Purification of Escherichia coli  RNase III Heterodimers......Page 160
Affinity chromatography......Page 161
References......Page 162
Characterizing Ribonucleases In Vitro: Examples of Synergies between Biochemical and Structural Analysis......Page 164
Introduction......Page 165
RNase II D209N Mutant......Page 166
Overexpression and Purification of RNase II and Its Derivatives......Page 167
RNase II exoribonucleolytic activity on poly(A) homopolymers......Page 170
Detection of ribonucleolytic activity on mRNA transcripts......Page 172
In vitro transcription reaction......Page 173
RNase II activity assay......Page 174
The usefulness of a set of RNA oligomers......Page 176
Electrophoretic mobility shift assay......Page 180
RNaseIID209N......Page 181
Filter-binding assays......Page 183
BIACORE: Surface plasmon resonance analysis......Page 188
Structural Studies of RNase II......Page 189
Acknowledgments......Page 190
References......Page 191
The Role of RNA Chaperone Hfq in Poly(A) Metabolism: Methods to Determine Positions, Abundance, and Lengths of Short Oligo(A) Tails......Page 194
Introduction......Page 195
Global Analysis of Polyadenylated Transcripts......Page 196
RT-PCR-Based Analysis of 3prime-RNA Ends......Page 197
A poly(A) targeted 3prime-RACE method to analyze 3prime-ends of oligoadenylated RNAs......Page 198
mRNA, anchors, and primers......Page 200
RNA extraction......Page 201
3prime-RACE amplification......Page 202
Locations of polyadenylation sites and lengths of the tails......Page 203
Hfq affects the frequency of oligoadenylated transcripts......Page 204
Oligo(A) tails are longer in the presence of Hfq......Page 205
A Method to Analyze Polyadenylation of Primary Transcripts......Page 207
Site-directed cleavage of rpsO mRNA by RNase H......Page 208
Northern blotting......Page 209
Conclusion......Page 210
References......Page 212
Assaying DEAD-box RNA Helicases and Their Role in mRNA Degradation in Escherichia coli......Page 215
Introduction......Page 216
The RNA-Dependent ATPase Activity of RhlB......Page 217
The ATPase-Dependent RNA Helicase Activity of RhlB......Page 219
The RhlB-PNPase mRNA Degradation Pathway......Page 220
Discussion and Perspective......Page 224
ATPase assay......Page 225
RNA unwinding assay......Page 226
mRNA degradation assay......Page 227
References......Page 228
Preparation of the Escherichia coli RNase E Protein and Reconstitution of the RNA Degradosome......Page 230
Introduction......Page 231
Buffers and solutions......Page 232
Growth of cultures......Page 233
Preparation of the AS-26 fraction......Page 234
Preparative gel electrophoresis......Page 235
Renaturation of Rne......Page 236
Nondenaturing purification of Rne......Page 237
Purification of the components......Page 238
Reconstitution......Page 239
References......Page 242
Identifying and Characterizing Substrates of the RNase E/G Family of Enzymes......Page 245
Introduction to E. coli RNase E and Its Homologs......Page 246
Mutant strains and the quenching of RNA metabolism......Page 249
Isolation of RNA and measurement of steady-state levels and half-lives......Page 250
Mapping the 5prime-ends of specific transcripts......Page 253
RNA ligase-mediated, reverse-transcription PCR assay......Page 254
Purification of RNase E and related enzymes......Page 258
Synthesis of transcripts with 5prime-monophosphorylated ends......Page 259
Cleavage conditions for E. coli RNase E and RNase G......Page 262
Investigation of recognition and cleavage with oligoribonucleotide substrates......Page 263
Summary......Page 265
References......Page 266
Construction and Characterization of E. coli K12 Strains in Which the Transcription of Selected Genes Is Desynchronized from Translation......Page 272
Introduction......Page 273
Procedure 1: Lysogenization of E. coli K12 with the lambdaDE3 phage encoding T7 RNAP......Page 275
Procedure 2: Introduction of the PT7-lacZ and Plac-lacZ cassettes in lambdaDE3 lysogens......Page 278
Procedure 3: Removal of the RNase E CTH......Page 280
beta-galactosidase assay......Page 281
RNA isolation......Page 283
Conclusion......Page 284
References......Page 285
Analysis of mRNA Decay in Bacillus subtilis......Page 288
Introduction......Page 289
Inhibition of B. subtilis Transcription......Page 292
RNA Isolation Protocol 1......Page 293
RNA Isolation Protocol 2......Page 294
RNA Isolation Protocol 3......Page 295
Labeled Size Marker for Small RNAs......Page 296
Northern-Blot Analysis Using Oligonucleotide Probes......Page 297
Mapping of 5prime- and 3prime-Ends......Page 298
Ribonuclease Mutant Strains......Page 300
References......Page 304
Assay of Bacillus subtilis Ribonucleases In Vitro......Page 306
RNase M5......Page 307
Purification of RNase M5......Page 308
Assay of RNase M5 activity in vitro......Page 309
Purification of EndoA......Page 310
Assay of EndoA activity in vitro......Page 312
RNase Z......Page 313
Purification of RNase P......Page 314
Holoenzyme reconstitution......Page 316
Assay of RNase P activity in vitro......Page 317
RNase J1......Page 318
Purification of RNase J1......Page 319
Assay of 5 to 3-exoribonuclease activity of RNase J1 in vitro......Page 320
Purification of RNase III......Page 321
YhcR......Page 323
Assay of YhcR activity in vitro......Page 324
Purification of PNPase......Page 325
RNase PH......Page 326
Purification of RNase PH......Page 327
Assay of RNase PH......Page 328
Assay of RNase R activity in vitro......Page 329
Assay of YhaM activity in vitro......Page 330
Assay of Nano-RNase activity in vitro......Page 331
Standard Protocol 1: T7 In Vitro Transcription Reactions......Page 332
Elution by pH gradient......Page 333
Standard Protocol 4: Native Purification of His-Tagged Proteins on BD-Talon......Page 334
References......Page 335
Staphylococcus aureus Endoribonuclease III: Purification and Properties......Page 338
Introduction......Page 339
S. aureus RNase III Is Not Essential for Cell Growth but Regulates Virulence Gene Expression......Page 341
Biochemical Properties and Substrate Specificity of S. aureus RNase III......Page 343
RNase III Overexpression and Purification......Page 346
RNA Substrate Preparation......Page 348
Mapping the RNase III Cleavage Sites In Vitro......Page 349
Denaturing Agarose Gel Electrophoresis and Northern Blotting......Page 351
Concluding Remarks......Page 352
References......Page 353
Studying tmRNA-Mediated Surveillance and Nonstop mRNA Decay......Page 357
Introduction......Page 358
Methodology......Page 362
Purification of SmpB protein......Page 363
Purification of tmRNA......Page 364
Affinity chromatography......Page 366
RNase R purification: Alternate procedure for obtaining RNA-free protein......Page 367
Purification of Lon......Page 368
Phage induction assays......Page 369
Endogenous tmRNAH6 tagging assay......Page 371
SmpB-tmRNA interactions with electrophoretic mobility shift assay......Page 372
Electrophoretic mobility shift assay......Page 373
Ribosome association assay......Page 374
Stalled ribosome enrichment assay......Page 375
In vitro RNA degradation assay......Page 376
Preparation of duplex RNA substrate and RNA decay assay......Page 378
RNA extraction......Page 379
In vivo GFP-ssrA protein stability assay......Page 381
In vivo lambda-CI-N protein stability assay......Page 382
GFP and GFP-ssrA purification......Page 383
References......Page 384
Analyses of mRNA Destabilization and Translational Inhibition Mediated by Hfq-Binding Small RNAs......Page 387
Overview......Page 388
Procedure......Page 390
Procedure......Page 392
Requirement of C-Terminal Scaffold Region of RNase E, Hfq, and SgrS......Page 393
Procedure......Page 394
Translational Repression Is the Primary Event for Silencing of ptsG mRNA by SgrS/Hfq/RNase E......Page 396
Procedure......Page 397
Base pairing Near Translation Initiation Region Is Crucial for SgrS Action......Page 398
Procedure......Page 400
Membrane Localization of ptsG mRNA Is Required for SgrS Action......Page 402
Conclusions......Page 403
References......Page 404
In Vivo and In Vitro Studies of RNA Degrading Activities in Archaea......Page 407
Introduction......Page 409
In vivo labeling of RNA and inhibition of transcription with actinomycin D......Page 410
RNA isolation from Archaea and Northern blot analysis......Page 412
Labeling and purification of substrates......Page 414
Thin-layer chromatography......Page 416
Isolation of RNA degrading activities by fractionation of S. solfataricus cell lysates......Page 417
Characterization of the recombinant RNA degrading dehydrogenases from S. solfataricus......Page 419
Purification of a protein with RNase activity from H. salinarum NRC-1 and its identification as aIF-5A......Page 420
Purification of recombinant haloarchaeal aIF-5A protein from E. coli......Page 422
Purification of the recombinant haloarchaeal aIF-5A protein from H. salinarum NRC-1......Page 423
Characterization of the endoribonucleolytic activity of the haloarchaeal aIF-5A......Page 426
Isolation and Characterization of a Protein Complex with Predicted Ribonuclease Activity from Sulfolobus solfataricus: The Archaeal Exosome......Page 427
Production of recombinant proteins and polyclonal antibodies......Page 428
Detection of putative subunits of the S. solfataricus exosome in high molecular weight fractions......Page 429
Purification of the exosome from S. solfataricus by coimmunoprecipitation and identification of its subunits......Page 430
Analysis of protein-protein interactions in vitro and reconstitution of the S. solfataricus exosome by refolding......Page 432
Functional characterization of reconstituted exosome complexes in vitro......Page 435
In vitro characterization of the native S. solfataricus exosome......Page 437
References......Page 439
Expression, Reconstitution, and Structure of an Archaeal RNA Degrading Exosome......Page 443
Introduction......Page 444
Cloning of the S. solfataricus exosome genes......Page 446
Overexpression of the archaeal exosome components......Page 447
Purification of archaeal exosome components......Page 448
In vitro reconstitution of archaeal exosome complexes......Page 449
Coexpression of archaeal exosome complexes......Page 450
Crystallization of apo S. solfataricus Rrp41/Rrp42 and Rrp4/Rrp41/Rrp42......Page 451
Structure determination of the Rrp41-Rrp42 complex......Page 452
RNA Binding Sites in the Archaeal Exosome......Page 454
RNA degradation experiments to probe the RNA access route......Page 455
Structures with 5-iodo-uridine RNA substrates to visualize the RNA access route......Page 457
References......Page 459
Polyadenylation-Mediated RNA Degradation in Plant Mitochondria......Page 462
Introduction......Page 463
Mutant plants affected in RNA degradation......Page 464
RNA extraction......Page 465
PCR amplification......Page 466
Comments on 3prime
-RACE......Page 468
Comments on cRT-PCR......Page 469
A cDNA library to identify polyadenylated substrates......Page 471
SMART cDNA library construction......Page 473
Comments on libraries of polyadenylated substrates......Page 474
Preparation of mitochondrial lysate......Page 475
In vitro degradation assays......Page 477
Analysis of degradation assays......Page 478
In organello expression systems......Page 479
Import and expression of the DNA construct......Page 480
Comments on in organello expression systems......Page 481
References......Page 482
In Vivo and In Vitro Approaches for Studying the Yeast Mitochondrial RNA Degradosome Complex......Page 485
Introduction......Page 486
Working with degradosome-deficient mutants......Page 488
Construction of degradosome-deficient deletant strains......Page 490
Preparation of yeast mitochondria......Page 491
Analysis of the steady-state RNA levels with Northern blotting......Page 492
Measuring mitochondrial transcription in organello......Page 493
Construction of the expression vectors......Page 494
Buffers......Page 495
Protocol......Page 496
Assaying the Ribonuclease Activity of the Degradosome and the Dss1 Protein......Page 497
Preparing the oligonucleotide substrates......Page 498
Gel assay......Page 499
Measuring reaction kinetics......Page 501
Measuring reaction kinetics......Page 502
Assaying the Helicase Activity of the Degradosome and the Suv3 Protein......Page 503
Unwinding assay......Page 504
Protein-RNA Binding Assay......Page 506
Concluding Remarks......Page 507
References......Page 508
Measuring mRNA Decay in Human Mitochondria......Page 511
Introduction......Page 512
Primer Design......Page 513
Cell Cultivation and RNA Extraction......Page 515
Polyadenylation Stabilizes Mitochondrial mRNAs......Page 516
Discussion......Page 518
References......Page 520
Detection and Characterization of Polyadenylated RNA in Eukarya, Bacteria, Archaea, and Organelles......Page 522
Introduction......Page 523
Oligo(dT) RT-PCR Detection of Polyadenylated Degradation Intermediates......Page 525
Steps 3 to 6: PCR amplification and gel extraction......Page 526
Steps 7 to 9: Cloning of the PCR products, selection, and sequencing analysis......Page 529
Circularized Reverse Transcription (cRT)-PCR-Sequencing/Labeling Method for the Characterization of Polyadenylated RNA......Page 530
Steps 1 and 2: RNA isolation, circularization, and RT......Page 531
Step 5a: Analysis by DNA sequencing......Page 533
Step 5b: Analysis by radioactive labeling......Page 534
Polyadenylation analysis by 3prime-end labeling and ribonuclease digestion......Page 537
References......Page 540
RNA Decay by Messenger RNA Interferases......Page 542
Introduction......Page 543
Plasmid construction......Page 544
His6-RelE expression and purification......Page 546
Synthesis of a test mRNA......Page 547
Development of a general mRNA probe vector plasmid......Page 548
Cell growth and RNA purification......Page 550
Primer extension analysis of lpp mRNA......Page 551
PCR sequencing......Page 552
Primer hybridization and reverse transcription......Page 554
References......Page 555
Author Index......Page 557
Subject Index......Page 575