Elsevier S & T

RNA Modification

Gott, Jonatha

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Table of contents
  • Contentsv
  • Contributorsxi
  • Prefacexv
  • Volumes in Seriesxvii
  • Section I: Modified Nucleotides1
  • Chapter 1: Identifying Modifications in RNA by MALDI Mass Spectrometry3
  • 1. Introduction4
  • 2. Experimental Strategy5
  • 3. Experimental Procedures7
  • 3.1. Isolation of RNA sequences7
  • 3.2. Digestion of RNA to oligonucleotides8
  • 3.3. MALDI mass spectrometry analysis9
  • 3.4. Tandem mass spectrometry13
  • 3.5. Comparison of MALDI and ESI techniques14
  • 4. Perspectives and Conclusion15
  • Acknowledgments17
  • References
  • Chapter 2: Identification of Modified Residues in RNAs by Reverse Transcription-Based Methods21
  • 1. Introduction22
  • 2. Reverse Transcription (RT)-Based Methods for Detection of Modified Residues24
  • 2.1. Reverse transcriptases used for RNA analysis26
  • 2.2. General protocol for primer labeling and reverse transcription of RNA27
  • 3. RNA Extraction from Various Cell Types29
  • 3.1. Protocol for the extraction of total RNA from archaeal or eukaryal cells29
  • 3.2. Protocol for the extraction of total RNA from yeast cells31
  • 4. RNA Modifications Detectable After Specific Chemical Treatment32
  • 4.1. Detection of inosine using glyoxal treatment followed by RNAse T1 hydrolysis32
  • 4.2. Detection of pseudouridine (C) residues36
  • 4.3. Protocol for the detection of pseudouridine residues in RNA using CMCT modification36
  • 4.4. Complementary approaches for C detection41
  • 4.5. Detection of 5-methylcytosine (m5C)41
  • 4.6. Detection of 7-methylguanine (m7G)42
  • 4.7. Detection of 20-O-methylated nucleotides using OH cleavage or 20-OH reactivity43
  • 4.8. Protocol for detection of 2'-O-methylated residues in RNA using OH-cleavage43
  • 4.9. Detection of 2'-O-methylated nucleotides by use of low dNTP concentrations44
  • 4.10. Protocol for detection of 2'-O-methylated residues in RNA by use of low dNTP concentrations45
  • 4.11. Other chemical modification methods specific for 20-O-methylations47
  • 4.12. Detection of dihydrouridine (D) residues by alkaline hydrolysis47
  • 5. RNA Modifications Leading to RT Pauses Without Preliminary Chemical Treatment48
  • 6. Conclusion48
  • References49
  • Section II: tRNA Modifications55
  • Chapter 3: Detection of Enzymatic Activity of Transfer RNA Modification Enzymes Using Radiolabeled t57
  • 1. Introduction59
  • 2. Identification of Modified Nucleotides in tRNA60
  • 3. Testing the Activity of RNA Modification Enzymes62
  • 3.1. In vitro production of T7-runoff transcripts and nearest-neighbor analysis of modified nucleoti63
  • 3.2. Experimental conditions for testing activity of tRNA modification enzymes in vitro68
  • 3.3. Optimization of enzymatic reaction conditions69
  • 4. Complete Digestion of RNA with Various Nucleases70
  • 4.1. Use of RNase T2 to generate 3'-monophosphate nucleosides70
  • 4.2. Homemade RNase mix71
  • 4.3. Use nuclease P1 to generate 50-monophosphate nucleosides72
  • 4.4. Use of venom phosphodiesterase VPD, eventually in combination with nuclease P1 to generate 5'-m72
  • 4.5. Use of piperidine to generate mixes of 20- and 30-monophosphate nucleosides73
  • 5. Analysis of RNA Digest Products by Thin-Layer Chromatography (TLC)73
  • 5.1. Preparation of the chromatography tanks and plates73
  • 5.2. First dimensional chromatography with solvent A (N1)75
  • 5.3. Second dimensional chromatography with solvent B (R2) or solvent C (N2)76
  • 5.4. Final identification of modified nucleotides on thin-layer plates77
  • 5.5. Quantification of modified nucleotides within an RNA fragment81
  • 6. Detection of Modified Nucleotides in Uniformly Labeled [32P]-RNA or [35S]-Containing RNA Extracte82
  • 6.1. Preparation of low-phosphate medium for culture of microorganisms in the presence of [32P]-orth82
  • 6.2. Preparation and purification of uniformly [32P]-labeled RNA83
  • 7. Base Composition Analysis of RNA by Postlabeling Procedures84
  • 7.1. Step 1: complete digestion of RNA (or RNA fragment) with RNase T285
  • 7.2. Step 2: [32P]-labeling of RNase T2 digest into 3',5'-diphosphate nucleosides with PNK85
  • 7.3. Step 3: hydrolysis of excess ATP with a mix of ATPase and ADPase (Apyrase)86
  • 7.4. Step 4: removal of 30-phosphate of diphosphate nucleosides with nuclease P187
  • 7.5. TLC analysis87
  • 8. RNA Sequencing of Purified Nonradiolabeled tRNA Species87
  • 9. Identification of Modified Residues in RNAs by Reverse Transcriptase-Based Methods90
  • 10. Discussion and Outlook92
  • Acknowledgments93
  • Note94
  • References94
  • Chapter 4: In Vitro Detection of the Enzymatic Activity of Folate-Dependent tRNA (Uracil-54,-C5)-M103
  • 1. Introduction104
  • 2. Overproduction and Purification of B subtilis tRNA (Uracil-54,-C5)-Methyltransferase106
  • 2.1. Expression plasmids and strains106
  • 2.2. Gene expression106
  • 2.3. Purification of the recombinant enzyme106
  • 3. Enzymatic Activity Assay107
  • 3.1. Reaction mix107
  • 3.2. Detection of m5U in tRNA109
  • 4. Phylogenetic Analysis110
  • 5. Discussion111
  • Acknowledgments116
  • References117
  • Chapter 5: Probing the Intermediacy of Covalent RNA Enzyme Complexes in RNA Modification Enzymes121
  • 1. Introduction122
  • 2. A Case Study for Probing Reaction Intermediacy: The Covalent RNA Complex of tRNA-Guanine Transgly124
  • 3. Denaturing Gel Electrophoresis: A Tool to Probe Enzyme-RNA Complexes125
  • 3.1. PAGE band-shift analysis of reaction mixtures126
  • 3.2. Band quantification by fluorescent detection127
  • 3.3. Detection of biotinylated-RNA containing bands by colorimetric assay128
  • 4. Analysis of the Chemical Competency of Covalent Enzyme-RNA Complexes132
  • 5. Analysis of the Rate of Formation of Covalent Enzyme-RNA Complexes133
  • 5.1. Kinetic studies that use rapid quench flow133
  • 6. Summary135
  • Acknowledgments136
  • References136
  • Chapter 6: Identification and Characterization of Modification Enzymes by Biochemical Analysis of th139
  • 1. Introduction140
  • 2. Methodology141
  • 3. Development of an Appropriate Biochemical.Assay141
  • 4. Parallel Biochemical Analysis of the Yeast Proteome with the MORF Library144
  • 5. Deconvolution of the Active Pool to Identify the Individual Gene Responsible for Activity147
  • 6. Confirmation of the Identity of the Gene Responsible for the Modification Activity147
  • 7. Possible Reasons for Lack of Success with this Approach149
  • 8. Conclusions150
  • References136
  • Chapter 7: Identification of Genes Encoding tRNA Modification Enzymes by Comparative Genomics153
  • 1. Introduction154
  • 2. Methods to Identify Missing tRNA Modification Genes155
  • 3. Homology-Based Genomic Data Mining Methods159
  • 4. Non-Homology-Based Genomic Data Mining Methods160
  • 4.1. Predictions based on gene clustering on chromosomes163
  • 4.2. Detecting protein fusion events167
  • 4.3. Searches based on phylogenetic distribution profiles169
  • 4.4. Mining other types of "Omics" data171
  • 4.5. Subsystem analysis172
  • 5. General Conclusion: The Power of Integration176
  • Acknowledgments176
  • References136
  • Chapter 8: Identification and Characterization of Archaeal and Fungal tRNA Methyltransferases185
  • 1. Introduction186
  • 2. Identification of tRNA Methyltransferases by Use of Genome Sequences187
  • 2.1. Cloning, expression, and protein purification192
  • 3. Substrates for Methyltransferase Assays192
  • 3.1. S-adenosylmethionine substrate192
  • 3.2. tRNA substrates194
  • 3.3. Protocol for small RNA purification from total RNA195
  • 3.4. In vitro transcripts196
  • 3.5. Protocol for in vitro transcription and cleavage of the hammerhead ribozyme197
  • 4. Methyltransferase Activity Assays198
  • 4.1. tRNA methyltransferase activity protocol198
  • 5. Identifying Modification Products200
  • 6. Identifying Modification Sites201
  • 6.1. Mapping modifications by primer extension202
  • 6.2. Primer extension protocol202
  • 7. tRNA Methyltransferases from M. jannaschii205
  • Acknowledgments205
  • References205
  • Chapter 9: Mass Spectrometric Identification and Characterization of RNA-Modifying Enzymes211
  • 1. Introduction212
  • 2. Ribonucleome Analysis: Quest for RNA-Modifying Genes212
  • 3. Strains Used for the Ribonucleome Analysis214
  • 4. Parallel Preparation of Total RNAs from E. coli or Yeast Strains214
  • 5. Isolation and Purification of Individual tRNAs from Yeast216
  • 6. Nucleoside Preparation217
  • 7. Mass Spectrometric Analysis of Total Nucleosides217
  • 8. LC/MS Profiling of Modified Nucleosides219
  • 9. RNase Digestion for RNA Fragment Analysis by LC/MS221
  • 10. Capillary LC NANO ESI/Mass Spectrometry221
  • 11. RNA Fragment Analysis by Capillary LC/MS223
  • 12. Discussion224
  • Acknowledgments227
  • References136
  • Further Reading229
  • Chapter 10: Chaplet Column Chromatography: Isolation of a Large Set of Individual RNAs in a Single S231
  • 1. Introduction231
  • 2. Outline of Chaplet Column Chromatography
  • 3. Materials and Reagents
  • 4. Preparation of Crude RNA Solution235
  • 5. Immobilization of DNA Probes
  • 6. RNA Isolation by the CCC Method237
  • 7. Discussion238
  • Acknowledgments238
  • References238
  • Section III: Sno-Mediated Modifications241
  • Chapter 11: Biochemical Purification of Box H/ACA RNPs Involved in Pseudouridylation243
  • 1. Introduction244
  • 2. Biotin-Streptavidin Affinity Purification Using Biotinylated 5FU-Containing RNA246
  • 2.1. Overview
  • 2.2. Synthesis of a biotinylated 5FU-containing U2 snRNA248
  • 2.3. Protocol248
  • 2.4. Protocol250
  • 3. TAP Tag Purification of Box H/ACA RNPs251
  • 3.1. Overview251
  • 3.2. Protocol253
  • 3.3. Protocol254
  • 3.4. Protocol255
  • 3.5. Protocol256
  • 4. Immunoprecipitation of Box H/ACA RNPs258
  • 4.1. Overview258
  • 4.2. Protocol259
  • Acknowledgments260
  • References
  • Chapter 12: In Vitro Reconstitution and Affinity Purification of Catalytically Active Archaeal Box C263
  • 1. Introduction264
  • 2. Cloning, Expression, and Preparation of M. jannaschii Box C/D sRNP Core Proteins265
  • 2.1. Cloning of M. jannaschii L7, Nop56/58, and fibrillarin genes265
  • 2.2. Recombinant core protein expression in bacterial cells266
  • 2.3. Affinity chromatographic isolation of L7 and fibrillarin core proteins267
  • 2.4. Isolation of Nop56/58 core protein by cation-exchange chromatography269
  • 3. Cloning and In Vitro Transcription of Archaeal Box C/D sRNAs270
  • 4. In Vitro Assembly of the M. jannaschii sR8 Box C/D sRNP Complex
  • 5. Assessment of In Vitro Assembled M. jannaschii Box C/D sRNP Methylation Activity271
  • 6. Sequential Affinity Chromatographic Purification of In Vitro Assembled Box C/D sRNPs272
  • 6.1. An overview272
  • 6.2. Affinity chromatography buffers276
  • 6.3. Preparation of the affinity chromatography resins276
  • 6.4. Assembly of the sR8 sRNP complex276
  • 6.5. Tandem affinity purification of in vitro assembled box C/D sRNP277
  • 7. Concluding Remarks280
  • Acknowledgments281
  • References281
  • Chapter 13: Identifying Effects of snoRNA-Guided Modifications on the Synthesis and.Function of the283
  • 1. Introduction284
  • 2. Experimental Strategies285
  • 2.1. The use of engineered snoRNAs to guide novel ribose methylations and pseudouridines in rRNA286
  • 2.2. Interfering with the activity of the catalytic snoRNP proteins287
  • 3. Analyzing snoRNA-Guided Modifications in Selected Regions of the Ribosome287
  • 3.1. Selecting modification sites for genetic depletion analysis288
  • 3.2. Design and construction of test strains290
  • 3.3. Analysis of growth and drug sensitivity294
  • 4. Determining Effects on rRNA and Ribosome Biogenesis297
  • 4.1. Monitoring rRNA processing by in vivo pulse-chase labeling298
  • 4.2. Determining the steady-state level of rRNA300
  • 4.3. Evaluating rRNA stability with in vivo labeling301
  • 5. Investigating Ribosomal Complexes302
  • 5.1. Polysome profiling302
  • 5.2. Distinguishing the elongating "80S" particle303
  • 5.3. Determining the 40S/60S ratio304
  • 6. Characterizing Ribosome Function304
  • 6.1. Analysis of the rate of protein synthesis304
  • 6.2. Analyzing translation fidelity305
  • 7. Chemical Probing of rRNA Structure306
  • 7.1. DMS modification306
  • 7.2. In vitro probing307
  • 7.3. Primer extension analysis307
  • 7.4. Primer extension sequencing308
  • Acknowledgments308
  • References308
  • Chapter 14: The U1 snRNA Hairpin II as a RNA Affinity Tag for Selecting snoRNP Complexes317
  • 1. Introduction318
  • 1.1. Why isolate snoRNPs?319
  • 1.2. Affinity tags used in RNP isolations320
  • 1.3. The rationale of the present RNA tagging scheme321
  • 1.4. The snoRNP substrates324
  • 2. Methodology326
  • 2.1. Overview of the method326
  • 2.2. Tagging the snoRNP components326
  • 2.3. Affinity enrichment of the processing snoRNPs U14 and U17329
  • 2.4. Other approaches for enriching snoRNPs with the U1hpII tag/U1A couple335
  • 2.5. Screening for interactions between snoRNPs338
  • 2.6. Potential limitations of these approaches340
  • 2.7. Other applications341
  • 3. Materials and Experimental Procedures342
  • 3.1. Yeast strains342
  • 3.2. TAP tagging of core proteins342
  • 3.3. Plasmid constructs342
  • 3.4. DNA oligonucleotides344
  • 3.5. Growth conditions345
  • 3.6. Affinity purification of snoRNPs346
  • 3.7. Screening for coselecting RNAs347
  • 3.8. RNA isolation and Northern analysis347
  • 3.9. Western analysis348
  • Acknowledgments348
  • References348
  • Chapter 15: A Dedicated Computational Approach for the Identification of Archaeal H/ACA sRNAs355
  • 1. Introduction356
  • 2. Method358
  • 2.1. Search for H/ACA-like motifs359
  • 2.2. Search for targets of the H/ACA-like motifs374
  • 2.3. Phylogenetic and experimental validation of the results382
  • 3. Conclusions383
  • References
  • Chapter 16: Reconstitution of Archaeal H/ACA sRNPs and Test of their Activity389
  • 1. Introduction390
  • 2. Overview of the Issues that can be Addressed by the Method393
  • 3. The Basic Principles of the Experiments393
  • 4. Materials and Reagents394
  • 4.1. Chemicals394
  • 4.2. General reagents394
  • 4.3. Equipment394
  • 4.4. Enzymes395
  • 4.5. Buffers395
  • 5. Methods396
  • 5.1. Production/purification of the archaeal proteins aCBF5, aNOP10, L7Ae, and aGAR1396
  • 5.2. Production of the box H/ACA guide sRNAs396
  • 5.3. Production of the RNA substrate398
  • 5.4. Assembly of box H/ACA sRNPs and their characterization by.EMSA399
  • 5.5. Analysis of the formation of an RNA duplex between the guide sRNA and the RNA substrate400
  • 5.6. Measurement of the RNA:Psi-synthase activity of the reconstituted sRNPs400
  • 5.7. Quantification of the data401
  • 6. General Comments403
  • 6.1. Choice of the size of the RNA substrate403
  • 6.2. No activity is detected403
  • Acknowledgments404
  • References
  • Author Index407
  • Subject Index427
Book details
  • Vendor Elsevier S & T
  • SKU 9780123741554
  • ISBN-13 9780080550916
  • Author Gott, Jonatha
  • Category Science
  • Subject Biochemistry

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The presence of modified nucleotides in cellular RNAs has been known for decades and over 100 distinct RNA modifications have been characterized to date. While the exact role of many of these modifications is still unclear, many are highly conserved across evolution and most contribute to the overall fitness of the organism. In recent years, new methods and bioinformatics approaches have been developed for the dissection of modification pathways and functions. These methods intersect a number of related fields, ranging from RNA processing to comparative genomics and systems biology. In addition, many of the techniques described in this volume have broad applicability, particularly in regards to the isolation, characterization, and reconstitution of ribonucleoprotein complexes, expanding the experimental repertoire available to all RNA researchers.