RNA Detection

Chapter 3

Identifying the m

6

A Methylome by Affinity Purification

and Sequencing

Phillip J. Hsu and Chuan He

Abstract

N^6 -methyladenosine (m^6 A) is the most abundant internal modification in eukaryotic mRNA, and is newly
emerging as a key posttranscriptional mRNA regulator. Recent research has uncovered insight into the
location and function of m^6 A sites on a large scale, in part due to the transcriptome-wide identification of
m^6 A sites by high-throughput sequencing (m^6 A-seq). Here, we present a protocol for m^6 A-seq, which
maps the m^6 A methylome by affinity purification and sequencing.

Key wordsN^6 -methyladenosine, Transcriptome, Methylome, Affinity purification, Sequencing

1 Introduction

Posttranscriptional modifications in eukaryotic messenger RNAs

(mRNAs) play a crucial role in fine-tuning gene expression in

response to changing conditions. Newly emerging as a key post-

transcriptional mRNA regulator isN^6 -methyladenosine (m^6 A), the

most abundant internal modification in mRNA [1]. m^6 A regulates

mRNA stability, localization, and translation; and is essential for life

[2–4]. Research on m^6 A, discovered over 4 decades ago, initially

advanced slowly due to lack of available methods. Previous meth-

ods to identify m^6 A sites were time-consuming and low-

throughput, relying on isolation of specific transcripts followed by

fragmentation to single nucleotides and biochemical analysis. Such

analytical methods had the capacity to analyze only several tran-

scripts at a time—much less the entire methylome. However, recent

advances in next-generation sequencing have brought about tech-

niques to gain insight into the location and function of m^6 A sites on

a large scale. In particular, m^6 A-seq, a high-throughput sequencing

method introduced in 2012 to accurately identify m^6 A sites

throughout the transcriptome, has begun to reveal the landscape

of the m^6 A methylome [5, 6].

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_3,©Springer Science+Business Media LLC 2018

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