RNA Modification - 1st Edition - ISBN: 9780128021927, 9780128023310

RNA Modification, Volume 560

1st Edition

Serial Volume Editors: Chuan He
eBook ISBN: 9780128023310
Hardcover ISBN: 9780128021927
Imprint: Academic Press
Published Date: 3rd August 2015
Page Count: 408
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Table of Contents

  • Preface
  • 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
  • Author Index
  • Subject Index

Description

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.

Key Features

  • 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

Readership

Scientists interested in RNA modifications, post-transcriptional gene expression regulation and regulatory RNA.


Details

No. of pages:
408
Language:
English
Copyright:
© Academic Press 2015
Published:
Imprint:
Academic Press
eBook ISBN:
9780128023310
Hardcover ISBN:
9780128021927

Reviews

Praise for the Series:
"Should be on the shelves of all libraries in the world as a whole collection." --Chemistry in Industry
"The work most often consulted in the lab." --Enzymologia
"The Methods in Enzymology series represents the gold-standard." --Neuroscience


About the Serial Volume Editors

Chuan He Serial Volume Editor

Chuan He, Ph.D., is the John T. Wilson Distinguished Service Professor in the Department of Chemistry and Director of the Institute for Biophysical Dynamics at the University of Chicago. He is also a joint Professor in the Department of Chemical Biology and Director of the Synthetic and Functional Biomolecules Center at Peking University. He was born in P. R. China in 1972 and received his B.S. (1994) from the University of Science and Technology of China. He received his Ph. D. degree from Massachusetts Institute of Technology in chemistry in 2000. After being trained as a Damon-Runyon postdoctoral fellow at Harvard University from 2000-2002, he joined the University of Chicago as an Assistant Professor, and was promoted to Associate Professor in 2008, Professor in 2010 and John T. Wilson Distinguished Service Professor in 2014. He is also a member of the Cancer Research Center at the University of Chicago. His research spans a broad range of chemical biology, epigenetics, cell biology, molecular biology, biochemistry, structural biology, and genomics. His recent research concerns reversible RNA and DNA methylation in biological regulation. His research group discovered the first RNA demethylase and showed that reversible RNA methylation significantly affects post-transcriptional gene expression regulation. He was selected as an Investigator of the Howard Hughes Medical Institute in 2013.

Affiliations and Expertise

University of Chicago