RNA Modification Volume 560 Methods in Enzymology 1st Edition by Chuan He 0128021926 9780128021927

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Product details:

ISBN 10: 0128021926 

ISBN 13: 9780128021927

Author: Chuan He

RNA Modification provides a useful examination of the science and its role in biological regulation, the current frontier of life science research, and includes various RNA modications and their role in gene expression. It represents the most up-to-date knowledge and protocols available today.

• Dynamic RNA modifications and their roles in biological regulation are the current frontier of life science research
• This volume of Methods in Enzymology represents up to date knowledge and protocols

Table of contents:

Chapter One: Methodology for the High-Throughput Identification and Characterization of tRNA Variants That Are Substrates for a tRNA Decay Pathway
Abstract
1 Introduction
2 High-Throughput Identification of tRNA Substrates Degraded by the RTD Pathway
3 Measurement of tRNA Levels of RTD Substrate SUP4oc Variants in the Presence of WT tRNATyr
4 Conclusions and Additional Applications of These Approaches
Acknowledgments

Chapter Two: Nucleoside Analysis by Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry
Abstract
1 Introduction
2 Nucleoside Preparation for LC/MS Analysis
3 HILIC/ESI-MS for Total Nucleosides
4 HILIC/ESI-MS Versus RPC/ESI-MS for Nucleoside Analysis
5 Profiling of Modified Nucleosides in HILIC/ESI-MS
6 Discussion
Acknowledgments

Chapter Three: A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA: Application to Stress-Induced Reprogramming of tRNA Modifications
Abstract
1 Introduction
2 Methods
3 Discussion
Acknowledgments

Chapter Four: Recognition of Specified RNA Modifications by the Innate Immune System
Abstract
1 Introduction
2 Preparation of tRNA Modivariants
3 Preparation and Stimulation of Human PBMC
4 Preparation of Human PBMCs
5 Stimulation of Human PBMC
Acknowledgment

Chapter Five: Kinetic Analysis of tRNA Methyltransferases
Abstract
1 Introduction
2 Methodology
3 Steady-State Assays
4 Pre-Steady-State Assays
5 Single-Turnover Assays
6 Conclusions
Acknowledgments

Chapter Six: Preparation of Human Nuclear RNA m6A Methyltransferases and Demethylases and Biochemical Characterization of Their Catalytic Activity
Abstract
1 Introduction
2 Expression of Human Nuclear RNA m6A Methyltransferase Core Complex METTL3–METTL14–WTAP in Insect Cell Expression System
3 Expression of Human Nuclear RNA m6A Demethylases FTO and ALKBH5
4 Biochemical Characterization of the Catalytic Activity of the m6A Methyltransferases and Demethylases
5 Conclusions
Acknowledgments

Chapter Seven: Transcriptome-Wide Mapping of N6-Methyladenosine by m6A-Seq
Abstract
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1: RNA Preparation and Fragmentation
6 Step 2: RNA Immunoprecipitation
7 Step 3: Library Preparation and Deep Sequencing
Acknowledgments

Chapter Eight: Probing RNA Modification Status at Single-Nucleotide Resolution in Total RNA
Abstract
1 Introduction
2 Methods
3 Notes
Acknowledgments

Chapter Nine: High-Resolution Mapping of N6-Methyladenosine in Transcriptome and Genome Using a Photo-Crosslinking-Assisted Strategy
Abstract
1 Introduction
2 Equipment
3 Materials
4 Protocol 1: Photo-Crosslinking-Assisted m6A Sequencing
5 Protocol 2: 6mA-CLIP-exo Sequencing
6 Conclusion
Acknowledgments

Chapter Ten: Pseudouridine in mRNA: Incorporation, Detection, and Recoding
Abstract
1 Introduction
2 Incorporation of Pseudouridine in TRM4 mRNA
3 Detection of Pseudouridine in the TRM4 mRNA
4 Recoding of Pseudouridylated PTC Codon
Acknowledgments

Chapter Eleven: Pseudo-Seq: Genome-Wide Detection of Pseudouridine Modifications in RNA
Abstract
1 Introduction
2 Sample Preparation and RNA Isolation
3 Pseudo-Seq Library Preparation
4 Pseudo-Seq Data Analysis
5 Experimental Considerations
6 Solutions, Reagents, and Common Protocols
Acknowledgments

Chapter Twelve: Pseudouridine Chemical Labeling and Profiling
Abstract
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1: Total RNA Isolation from Mammalian Tissues and Cells
6 Step 2: mRNA Isolation
7 Step 3: N3-CMC Labeling and Click Reaction
8 Step 4: Enrich ψ-Containing RNA Fragments by Streptavidin Pull Down
9 Step 5: RNA Ligation
10 Step 6: Reverse Transcription
11 Step 7: Circligation, Linearization, and PCR Amplification

Chapter Thirteen: Experimental Approaches for Target Profiling of RNA Cytosine Methyltransferases
Abstract
1 Introduction
2 Methods

Chapter Fourteen: RNA 5-Methylcytosine Analysis by Bisulfite Sequencing
Abstract
1 Introduction
2 Methods for Identifying m5C-Modified RNA in Sequence Context
3 Considerations Before RNA Bisulfite Sequencing (RNA-BisSeq)
4 General Protocol for RNA-BisSeq
5 RNA-BisSeq Data Analysis
6 Limitations of RNA-BisSeq
Acknowledgments

Chapter Fifteen: Biochemical and Transcriptome-Wide Identification of A-to-I RNA Editing Sites by ICE-Seq
Abstract
1 Introduction
2 Outline of the ICE-seq Protocol
3 Inosine Cyanoethylation with Acrylonitrile
4 cDNA Library Preparation and Deep Sequencing
5 Data Analysis
6 Evaluation of ICE-Seq
Acknowledgments

Chapter Sixteen: Radical SAM-Mediated Methylation of Ribosomal RNA
Abstract
1 Introduction
2 Mechanism of RNA Methylation by RlmN and Cfr
3 Expression of RlmN and Cfr
4 Purification of RlmN and Cfr
5 In Vitro Methylation Assays for RlmN and Cfr
6 In Vivo Activity Assay for RlmN and Cfr
7 Summary and Concluding Remarks
Acknowledgments

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Tags: Chuan He, RNA, Modification, Methods,