RNA Modification 1st Edition by Jonatha Gott ISBN 0123741556 9780123741554

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ISBN 10: 0123741556 
ISBN 13: 9780123741554
Author: Jonatha Gott

RNA Modification 1st Table of contents:

Section I: Modified Nucleotides
Chapter 1: Identifying Modifications in RNA by MALDI Mass Spectrometry
 1. Introduction
 2. Experimental Strategy
 3. Experimental Procedures
  3.1. Isolation of RNA sequences
  3.2. Digestion of RNA to oligonucleotides
  3.3. MALDI mass spectrometry analysis
  3.4. Tandem mass spectrometry
  3.5. Comparison of MALDI and ESI techniques
 4. Perspectives and Conclusion
 Acknowledgments
 References

Chapter 2: Identification of Modified Residues in RNAs by Reverse Transcription-Based Methods
 1. Introduction
 2. Reverse Transcription (RT)-Based Methods for Detection of Modified Residues
  2.1. Reverse transcriptases used for RNA analysis
  2.2. General protocol for primer labeling and reverse transcription of RNA
 3. RNA Extraction from Various Cell Types
  3.1. Protocol for the extraction of total RNA from archaeal or eukaryal cells
  3.2. Protocol for the extraction of total RNA from yeast cells
 4. RNA Modifications Detectable After Specific Chemical Treatment
  4.1. Detection of inosine using glyoxal treatment followed by RNAse T1 hydrolysis
  4.2. Detection of pseudouridine (Ψ) residues
  4.3. Protocol for the detection of pseudouridine residues in RNA using CMCT modification
  4.4. Complementary approaches for Ψ detection
  4.5. Detection of 5-methylcytosine (m5C)
  4.6. Detection of 7-methylguanine (m7G)
  4.7. Detection of 2′-O-methylated nucleotides using OH cleavage or 2′-OH reactivity
  4.8. Protocol for detection of 2′-O-methylated residues in RNA using OH-cleavage
  4.9. Detection of 2′-O-methylated nucleotides by use of low dNTP concentrations
  4.10. Protocol for detection of 2′-O-methylated residues in RNA by use of low dNTP concentrations
  4.11. Other chemical modification methods specific for 2′-O-methylations
  4.12. Detection of dihydrouridine (D) residues by alkaline hydrolysis
 5. RNA Modifications Leading to RT Pauses Without Preliminary Chemical Treatment
 6. Conclusion
 References

Section II: tRNA Modifications
Chapter 3: Detection of Enzymatic Activity of Transfer RNA Modification Enzymes Using Radiolabeled tRNA
 1. Introduction
 2. Identification of Modified Nucleotides in tRNA
 3. Testing the Activity of RNA Modification Enzymes
  3.1. In vitro production of T7-runoff transcripts and nearest-neighbor analysis of modified nucleotides
  3.2. Experimental conditions for testing activity of tRNA modification enzymes in vitro
  3.3. Optimization of enzymatic reaction conditions
 4. Complete Digestion of RNA with Various Nucleases
  4.1. Use of RNase T2 to generate 3′-monophosphate nucleosides
  4.2. Homemade RNase mix
  4.3. Use nuclease P1 to generate 5′-monophosphate nucleosides
  4.4. Use of venom phosphodiesterase VPD, eventually in combination with nuclease P1
  4.5. Use of piperidine to generate mixes of 2′- and 3′-monophosphate nucleosides
 5. Analysis of RNA Digest Products by Thin-Layer Chromatography (TLC)
  5.1. Preparation of the chromatography tanks and plates
  5.2. First dimensional chromatography with solvent A (N1)
  5.3. Second dimensional chromatography with solvent B (R2) or solvent C (N2)
  5.4. Final identification of modified nucleotides on thin-layer plates
  5.5. Quantification of modified nucleotides within an RNA fragment
 6. Detection of Modified Nucleotides in Uniformly Labeled [32P]-RNA or [35S]-Containing RNA Extracts
  6.1. Preparation of low-phosphate medium for culture of microorganisms in the presence of [32P]-orthophosphate
  6.2. Preparation and purification of uniformly [32P]-labeled RNA
 7. Base Composition Analysis of RNA by Postlabeling Procedures
  7.1. Complete digestion of RNA with RNase T2
  7.2. [32P]-labeling of RNase T2 digest into 3′,5′-diphosphate nucleosides with PNK
  7.3. Hydrolysis of excess ATP with a mix of ATPase and ADPase (Apyrase)
  7.4. Removal of 3′-phosphate of diphosphate nucleosides with nuclease P1
  7.5. TLC analysis
 8. RNA Sequencing of Purified Nonradiolabeled tRNA Species
 9. Identification of Modified Residues in RNAs by Reverse Transcriptase-Based Methods
 10. Discussion and Outlook
 Acknowledgments
 Note
 References

Chapter 4: In Vitro Detection of the Enzymatic Activity of Folate-Dependent tRNA (Uracil-54,-C5)-Methyltransferase
 1. Introduction
 2. Overproduction and Purification of B. subtilis tRNA (Uracil-54,-C5)-Methyltransferase
  2.1. Expression plasmids and strains
  2.2. Gene expression
  2.3. Purification of the recombinant enzyme
 3. Enzymatic Activity Assay
  3.1. Reaction mix
  3.2. Detection of m5U in tRNA
 4. Phylogenetic Analysis
 5. Discussion
 Acknowledgments
 References

Chapter 5: Probing the Intermediacy of Covalent RNA Enzyme Complexes in RNA Modification Enzymes
 1. Introduction
 2. A Case Study for Probing Reaction Intermediacy: The Covalent RNA Complex of tRNA-Guanine Transglycosylase
 3. Denaturing Gel Electrophoresis: A Tool to Probe Enzyme-RNA Complexes
  3.1. PAGE band-shift analysis of reaction mixtures
  3.2. Band quantification by fluorescent detection
  3.3. Detection of biotinylated-RNA containing bands by colorimetric assay
 4. Analysis of the Chemical Competency of Covalent Enzyme-RNA Complexes
 5. Analysis of the Rate of Formation of Covalent Enzyme-RNA Complexes
  5.1. Kinetic studies using rapid quench flow

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