Main description:

This detailed book describes some of the most recent advances and up-to-date methodologies to detect, quantify, analyze, and elucidate the biological function of different types of RNA modifications. Importantly, the methodologies and tools described herein can be applied to a wide variety of organisms and can be used to address biological and clinical questions. Beginning with a section on bioinformatics tools, the collection continues with sections on detecting RNA modifications using Nanopore direct RNA sequencing, next-generation sequencing approaches, qPCR- and molecular biology-based methods, mass spectrometry- and NMR-based methods, as well as approaches to assess kinetics, determinants, and functions of RNA modifications. Written for the highly successful Methods in Molecular Biology series style, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, RNA Modifications: Methods and Protocols serves as an ideal guide for those working directly in the fields of epitranscriptomics and post-transcriptional gene regulation, as well as for scientists and clinicians interested in bioinformatic tools to study RNA modifications and techniques to dissect their roles in physiology and disease.
Chapter 20 is available open access under a CC BY 4.0 license.

Contents:

Part I: Bioinformatic Tools to Study RNA Modifications

1. RNA Post-Transcriptional Modification Mapping Data Analysis Using RNA Framework

Ilaria Manfredonia and Danny Incarnato

2. An Informatics Pipeline for Profiling and Annotating RNA Modifications

Qi Liu, Xiaoqiang Lang, and Richard I. Gregory

Part II: Detecting RNA Modifications Using Nanopore Direct RNA Sequencing

3. EpiNano: Detection of m6A RNA Modifications Using Oxford Nanopore Direct RNA Sequencing

Huanle Liu, Oguzhan Begik, and Eva Maria Novoa

4. Adaptation of Human Ribosomal RNA for Nanopore Sequencing of Canonical and Modified Nucleotides

Miten Jain, Hugh E. Olsen, Mark Akeson, and Robin Abu-Shumays

Part III: Next-Generation Sequencing Approaches to Detect and Capture Modified RNAs

5. AlkAniline-Seq: A Highly Sensitive and Specific Method for Simultaneous Mapping of 7-Methyl-Guanosine (m7G) and 3-Methyl-Cytidine (m3C) in RNAs by High-Throughput Sequencing

Virginie Marchand, Lilia Ayadi, Valerie Bourguignon-Igel, Mark Helm, and Yuri Motorin

6. Transcriptome-Wide Detection of Internal N7-Methylguanosine

Li-Sheng Zhang, Chang Liu, and Chuan He

7. miCLIP-MaPseq Identifies Substrates of Radical SAM RNA Methylating Enzyme Using Mechanistic Crosslinking and Mismatch Profiling

Vanja Stojkovic, David E. Weinberg, and Danica Galonic Fujimori

8. Mapping RNA Modifications Using Photo-Crosslinking-Assisted Modification Sequencing

Bryan R. Cullen and Kevin Tsai

9. Quantitative and Single Nucleotide Resolution Profiling of RNA 5-Methylcytosine

Jun Li, Xingyu Wu, Trung Do, Vy Nguyen, Jing Zhao, Pei Qin Ng, Alice Burgess, Rakesh David, and Iain Searle

10. A Small RNA-Seq Protocol with Less Bias and Improved Capture of 2'-O-Methyl RNAs

Erwin L. van Dijk and Claude Thermes

Part IV: Assessing RNA Modifications Using qPCR- and Molecular Biology-Based Methods

11. Assessing 2'-O-Methylation of mRNA Using Quantitative PCR

Brittany A. Elliott and Christopher L. Holley

12. Relative Quantification of Residue Specific m6A RNA Methylation Using m6A-RT-QPCR

Ane Olazagoitia-Garmendia and Ainara Castellanos-Rubio

13. Monitoring the 5-Methoxycarbonylmethyl-2-Thiouridine (mcm5s2U) Modification Utilizing the Gamma-Toxin Endonuclease

Jenna M. Lentini and Dragony Fu

14. Analysis of Queuosine tRNA Modifications Using APB Northern Blot Assays

Cansu Cirzi and Francesca Tuorto

15. Detecting ADP-Ribosylation in RNA

Deeksha Munnur and Ivan Ahel

Part V: Mass Spectrometry- and NMR-Based Methods for RNA Modifications Analysis

16. Detecting Internal N7-Methylguanosine mRNA Modifications by Differential Enzymatic Digestion Coupled with Mass Spectrometry Analysis

Xue-Jiao You and Bi-Feng Yuan

17. A General LC-MS-Based Method for Direct and De Novo Sequencing of RNA Mixtures Containing Both Canonical and Modified Nucleotides

Ning Zhang, Shundi Shi, Xiaohong Yuan, Wenhao Ni, Xuanting Wang, Barney Yoo, Tony Z. Jia, Wenjia Li, and Shenglong Zhang

18. Quantification of Modified Nucleosides in the Context of NAIL-MS

Matthias Heiss, Kayla Borland, Yasemin Yoluc, and Stefanie Kellner

19. A Method to Monitor the Introduction of Post-Transcriptional Modifications in tRNAs with NMR Spectroscopy

Alexandre Gato, Marjorie Catala, Carine Tisne, and Pierre Barraud

Part VI: Approaches to Assess Kinetics, Determinants, and Functions of RNA Modifications

20. Effects of mRNA Modifications on Translation: An Overview

Bijoyita Roy

21. Assaying the Molecular Determinants and Kinetics of RNA Pseudouridylation by H/ACA snoRNAs and Stand-Alone Pseudouridine Synthases

Dominic P. Czekay, Sarah K. Schultz, and Ute Kothe

22. Investigating Pseudouridylation Mechanisms by High-Throughput In Vitro RNA Pseudouridylation and Sequencing

Nicole M. Martinez and Wendy V. Gilbert

23. Targeted RNA m6A Editing Using Engineered CRISPR-Cas9 Conjugates

Xiao-Min Liu and Shu-Bing Qian