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Illustrating the Epitranscriptome at Nucleotide Resolution Using Methylation-iCLIP (miCLIP)

  • Harry George
  • Jernej Ule
  • Shobbir HussainEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)

Abstract

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.”

Key words

Epitranscriptome Epitranscriptomics RNA methylation Methylation-iCLIP miCLIP NSun2 

Notes

Acknowledgments

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 methylation-iCLIP. 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).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Biology and BiochemistryUniversity of BathBathUK
  2. 2.Department of Molecular NeuroscienceUniversity College LondonLondonUK

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