Save up to 30% on Elsevier print and eBooks with free shipping. No promo code needed.
Save up to 30% on print and eBooks.
RNA Modification
1st Edition, Volume 560 - August 3, 2015
Editor: Chuan He
Language: English
Hardback ISBN:9780128021927
9 7 8 - 0 - 1 2 - 8 0 2 1 9 2 - 7
eBook ISBN:9780128023310
9 7 8 - 0 - 1 2 - 8 0 2 3 3 1 - 0
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 modic…Read more
Purchase options
LIMITED OFFER
Save 50% on book bundles
Immediately download your ebook while waiting for your print delivery. No promo code is needed.
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
Scientists interested in RNA modifications, post-transcriptional gene expression regulation and regulatory RNA.
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
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
No. of pages: 408
Language: English
Edition: 1
Volume: 560
Published: August 3, 2015
Imprint: Academic Press
Hardback ISBN: 9780128021927
eBook ISBN: 9780128023310
CH
Chuan He
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.