Illustrating the epitranscriptome at nucleotide resolution using methylation-iCLIP (miCLIP)

TY - CHAP

T1 - Illustrating the epitranscriptome at nucleotide resolution using methylation-iCLIP (miCLIP)

AU - George, Harry

AU - Ule, Jernej

AU - Hussain, Shobbir

N1 - Funding Information: We wish to acknowledge Dr. Julian Konig who codeveloped the original iCLIP protocol, and Dr. Yoichiro Sugimoto for helpful feedback and discussions during the development of methylationiCLIP. Research in the SH laboratory is supported by a Seed Award in Science from the Wellcome Trust (WT108285MA), and a Responsive Mode Project Grant from the Biotechnology and Biosciences Research Council (BBSRC) UK (BB/N000749/1). Publisher Copyright: © Springer Science+Business Media LLC 2017.

PY - 2017/10/7

Y1 - 2017/10/7

N2 - Next-generation sequencing technologies have enabled the transcriptome to be profiled at a previously unprecedented speed and depth. This yielded insights into fundamental transcriptomic processes such as gene transcription, RNA processing, and mRNA splicing. Immunoprecipitation-based transcriptomic methods such as individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) have also allowed high-resolution analysis of the RNA interactions of a protein of interest, thus revealing new regulatory mechanisms. We and others have recently modified this method to profile RNA methylation, and we refer to this customized technique as methylation-iCLIP (miCLIP). Variants of miCLIP have been used to map the methyl-5-cytosine (m5C) or methyl-6-adenosine (m6A) modification at nucleotide resolution in the human transcriptome. Here we describe the m5C-miCLIP protocol, discuss how it yields the nucleotide-resolution RNA modification maps, and comment on how these have contributed to the new field of molecular genetics research coined “epitranscriptomics”.

AB - Next-generation sequencing technologies have enabled the transcriptome to be profiled at a previously unprecedented speed and depth. This yielded insights into fundamental transcriptomic processes such as gene transcription, RNA processing, and mRNA splicing. Immunoprecipitation-based transcriptomic methods such as individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) have also allowed high-resolution analysis of the RNA interactions of a protein of interest, thus revealing new regulatory mechanisms. We and others have recently modified this method to profile RNA methylation, and we refer to this customized technique as methylation-iCLIP (miCLIP). Variants of miCLIP have been used to map the methyl-5-cytosine (m5C) or methyl-6-adenosine (m6A) modification at nucleotide resolution in the human transcriptome. Here we describe the m5C-miCLIP protocol, discuss how it yields the nucleotide-resolution RNA modification maps, and comment on how these have contributed to the new field of molecular genetics research coined “epitranscriptomics”.

KW - Epitranscriptome

KW - Epitranscriptomics

KW - Methylation-iCLIP

KW - MiCLIP

KW - NSun2

KW - RNA methylation

UR - http://www.scopus.com/inward/record.url?scp=85016934404&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-6807-7_7

DO - 10.1007/978-1-4939-6807-7_7

M3 - Chapter

C2 - 28349456

AN - SCOPUS:85016934404

T3 - Methods in Molecular Biology

SP - 91

EP - 106

BT - Methods in Molecular Biology

PB - Humana Press Inc

ER -