title: Illustrating the Epitranscriptome at Nucleotide Resolution Using Methylation-iCLIP (miCLIP).
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."
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK.
authors.forename: Harry
authors.initials: H
authors.lastname: George
authors.affiliation: Department of Molecular Neuroscience, University College London, London, WC1N 3BG, UK.
authors.forename: Jernej
authors.initials: J
authors.lastname: Ule
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK. s.hussain@bath.ac.uk.
authors.forename: Shobbir
authors.initials: S
authors.lastname: Hussain
mesh_terms: D002460:Cell Line; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods; D020869:Gene Expression Profiling; D015723:Gene Library; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007553:Isotope Labeling; D008745:Methylation; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D059467:Transcriptome
chemical_list: D012313:RNA
keywords: Epitranscriptome; Epitranscriptomics; Methylation-iCLIP; NSun2; RNA methylation; miCLIP