Illustrating the Epitranscriptome at Nucleotide Resolution Using Methylation-iCLIP (miCLIP)
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 wordsEpitranscriptome Epitranscriptomics RNA methylation Methylation-iCLIP miCLIP NSun2
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).