RNA Detection

Chapter 2

Quantification of 2
0

• O-Me Residues in RNA Using

Next-Generation Sequencing (Illumina RiboMethSeq

Protocol)

Lilia Ayadi, Yuri Motorin, and Virginie Marchand

Abstract

RNA 2^0 - O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from
bacteria, archaea, and eukarya. We and others have recently published accurate and sensitive detection of
these modifications on native RNA at a single base resolution by high-throughput sequencing technologies.
Relative quantification of these modifications is still under progress and would probably reduce the number
of false positives due to 3D RNA structure. Therefore, here, we describe a reliable and optimized protocol
for quantification of 2^0 - O-Methylations based on alkaline fragmentation of RNA coupled to a commonly
used ligation approach followed by Illumina sequencing. For this purpose, we describe how to prepare
in vitro transcribed yeast 18S and 25S rRNA used as a reference for unmodified rRNAs and to compare
them to purified 18S and 25S rRNA from yeast total RNA preparation. These reconstructed rRNA mixes
were combined at different ratios and processed for RiboMethseq protocol.
This technique will be applicable for routine parallel treatment of biological and clinical samples to
decipher the functions of 2^0 - O-methylations in normal and pathologic processes or during development.

Key words 20 -O-methylation, High-throughput sequencing, RNA modification, Ribose methyla-

tion, Alkaline fragmentation

1 Introduction

Modulation of RNA properties by posttranscriptional mechanisms

of RNA modification is a newly discovered layer for regulation of

gene expression. At the level of epitranscriptome, these dynamic

and regulated RNA modification events contribute to RNA-RNA

and RNA-protein interactions, modulate alternative splicing,

mRNA translation, RNA transport and localization [1, 2]. The

current major challenge in the field is a careful mapping of different

RNA modifications in coding and noncoding RNAs, as well as

precise quantification of the modification rate for every given site.

Taking into account that at least thousands of RNA modified

nucleotides expected to be present in higher eukaryotic

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_2,©Springer Science+Business Media LLC 2018

29