RNA Modification Enzymes

1st Edition, Volume 658 - September 11, 2021
Imprint: Academic Press
Editor: Jane E. Jackman
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 8 2 3 5 8 5 - 0
eBook ISBN:
9 7 8 - 0 - 1 2 - 8 2 3 5 8 6 - 7

RNA Modification Enzymes, Volume 659 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on a variet… Read more

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RNA Modification Enzymes, Volume 659 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on a variety of related topics, including Locating chemical modifications in RNA sequences through ribonucleases and LC-MS based analysis, Development of RNA modification mapping pipelines for high-throughput sequencing approaches, AlkAniline-Seq for high-resolution mapping RNA m7G and m3C modifications, Facile detection of RNA phospho-methylation in cells, Detection and analysis of glycosylated queuosine modifications, A comprehensive pipeline for analysis of RNA 3’-end modification, Analysis of the epitranscriptome with ion-pairing reagent free oligonucleotide mass spectrometry, and more.

Cover image
Title page
Table of Contents
Copyright
Contributors
Preface
Chapter One: Locating chemical modifications in RNA sequences through ribonucleases and LC-MS based analysis
Abstract
1: Introduction
2: Characterization of chemical modifications in RNA
3: Tools to characterize modified RNA sequence
4: Protocols
5: Summary
Acknowledgment
Chapter Two: Mapping of 7-methylguanosine (m7G), 3-methylcytidine (m3C), dihydrouridine (D) and 5-hydroxycytidine (ho5C) RNA modifications by AlkAniline-Seq
Abstract
1: Introduction
2: Chemical approaches for mapping of m7G, m3C, D and ho5C
3: Overview of AlkAniline-Seq protocol
4: Analysis of tRNA and rRNA in total RNA fraction
5: AlkAniline-Seq protocol
6: Data analysis and interpretation
7: Limitations
8: Summary
Acknowledgments
Chapter Three: Facile detection of RNA phospho-methylation in cells and tissues
Abstract
1: Introduction
2: Protocol
3: Expected outcomes
4: Quantification and statistical analysis
5: Advantages
6: Limitations
7: Optimization and troubleshooting
8: Safety considerations and standards
9: Alternative methods/procedures
Chapter Four: Quantitative probing of glycosylated queuosine modifications in tRNA
Abstract
1: Introduction
2: Methods
3: Notes
Acknowledgments
Chapter Five: CTS tag-based methods for investigating mitochondrial RNA modification factors in Trypanosoma brucei
Abstract
1: Introduction
2: Protein affinity purification and in vivo proximity labeling
3: UV-crosslinking tandem affinity purification sequencing (CTAP-SEQ)
4: Immunofluorescence imaging of CTS-tagged proteins
5: Summary
Acknowledgments
Chapter Six: Analysis of the epitranscriptome with ion-pairing reagent free oligonucleotide mass spectrometry
Abstract
1: Introduction
2: Materials
3: Methods
4: Notes
Acknowledgments
Chapter Seven: RNA immunoprecipitation to identify in vivo targets of RNA editing and modifying enzymes
Abstract
1: Introduction
2: Factors to consider when designing a RIP assay
3: Step-by-step method details
4: Troubleshooting
5: Summary
Acknowledgment
Chapter Eight: Chemoenzymatic labeling of RNA to enrich, detect and identify methyltransferase-target sites
Abstract
1: Before you begin
2: Key resources table
3: Materials and equipment
4: Step-by-step method details
5: Expected outcomes
6: Quantification and data analysis
7: Advantages
8: Limitations
9: Optimization and troubleshooting
10: Safety considerations and standards
11: Alternative methods/procedures
Acknowledgments
Chapter Nine: Analysis of codon-specific translation by ribosome profiling
Abstract
1: Introduction
2: Before you begin
3: Step-by-step method details
4: Expected outcomes
5: Quantification and statistical analysis
6: Advantages
7: Limitations
8: Optimization and troubleshooting
9: Safety considerations and standards
10: Alternative methods/procedures
Chapter Ten: Partially modified tRNAs for the study of tRNA maturation and function
Abstract
1: Introduction
2: Partial modification of in vitro transcribed tRNA
3: tRNA modification activity assays with partially modified tRNAs
4: Determining tRNA affinity with the nitrocellulose filtration assay
5: Future perspectives
Acknowledgments
Chapter Eleven: Transient kinetic analysis for studying ionizations in RNA modification enzyme mechanisms
Abstract
1: Introduction
2: Before you begin
3: Key resources table
4: Materials and equipment
5: Step-by-step method details
6: Preparation of site-specifically labeled tRNA
7: Preparation of uniformly labeled tRNAs
8: Determining pKa for enzyme reactions using single turnover assays
9: Application of pH-rate profiles to study bifunctional tRNA methyltransferases
10: Conclusions
Chapter Twelve: Pseudouridine site assignment by high-throughput in vitro RNA pseudouridylation and sequencing
Abstract
1: Introduction
2: Pool design
3: Materials and equipment
4: Protocol
5: Quantification and statistical analysis
6: Alternative methods/procedures
7: Advantages
8: Limitations
9: Conclusions
Acknowledgments
Chapter Thirteen: Detection of tRNA-specific adenosine deaminase activity and wobble inosine modification in human cell lysates
Abstract
1: Introduction
2: Preparation of human cell extracts
3: In vitro transcription of internally radiolabeled tRNA
4: Detection of wobble adenosine deaminase activity in human cell lysates
5: Separation of nucleotides by thin layer chromatography
6: RT-PCR of tRNAs for cDNA sequencing
Acknowledgments
Chapter Fourteen: In vitro and in cellula site-directed RNA editing using the λNDD-BoxB system
Abstract
1: Introduction
2: λNDD-BoxB system
3: Construct design
4: In vitro editing assay
5: In cellula editing assay
6: Conclusion
Acknowledgments
Chapter Fifteen: A semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes
Abstract
1: Introduction
2: Description of semi-quantitative pull-down methodology for Trm140 binding to tRNA substrates
3: Conclusions, additional applications and experimental concerns
Chapter Sixteen: Investigating the consequences of mRNA modifications on protein synthesis using in vitro translation assays
Abstract
1: Introduction
2: Ribosome purification
3: Translation factor purification
4: Purification of natively modified tRNA
5: Preparing aminoacylated tRNAs and mRNA
6: Initiation complex formation and amino acid addition reactions
7: Miscoding screening assays
8: Measuring rate constants for miscoding
9: Quantification and kinetic analysis
Acknowledgments
Chapter Seventeen: Mass spectrometric analysis of mRNA 5′ terminal modifications
Abstract
1: Introduction
2: Preparation of mRNA 5′-terminal fragments
3: Mass spectrometry
4: Data analysis
5: Discussion
Acknowledgments
Chapter Eighteen: CLIP-Seq to identify targets and interactions of RNA binding proteins and RNA modifying enzymes
Abstract
1: Introduction
2: Equipment
3: Materials
4: Solutions/buffers
5: Protocol
6: Step 1. Expression of protein of interest
7: Step 2. UV crosslinking
8: Step 3. Lysate preparation
9: Step 4. Magnetic bead preparation
10: Step 5. Pulldown of RNA-protein complex
11: Step 6. Quality control
12: Step 7. Library construction
13: Step 8. Library quality control
14: Step 9. Sequencing
15: Limitations
16: Alternative methods
Acknowledgments
Chapter Nineteen: CRISPR mediated genome editing, a tool to dissect RNA modification processes
Abstract
1: Introduction
2: Creation of plasmid and repair template to facilitate CRISPR-Cas9 mutagenesis
3: C. albicans transformation and mutation verification
4: Conclusions and variations of these approaches
Key resources

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RNA Modification Enzymes

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