title: iCLIP: protein-RNA interactions at nucleotide resolution.
abstract: RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK; MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Ina
authors.initials: I
authors.lastname: Huppertz
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK; MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Jan
authors.initials: J
authors.lastname: Attig
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK; MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Andrea
authors.initials: A
authors.lastname: D'Ambrogio
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Easton
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK; MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Sibley
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Sugimoto
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK; Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia.
authors.forename: Mojca
authors.initials: M
authors.lastname: Tajnik
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK; MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK; Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany. Electronic address: j.koenig@imb-mainz.de.
authors.forename: Julian
authors.initials: J
authors.lastname: König
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK; MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: j.ule@ucl.ac.uk.
authors.forename: Jernej
authors.initials: J
authors.lastname: Ule
mesh_terms: D001665:Binding Sites; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics; D005786:Gene Expression Regulation; D015723:Gene Library; D006367:HeLa Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D014466:Ultraviolet Rays
chemical_list: D004270:DNA, Circular; D016601:RNA-Binding Proteins; D012313:RNA
keywords: High-throughput sequencing; Post-transcriptional regulation; Protein–RNA interaction; RNA; RNA-binding protein; UV crosslinking and immunoprecipitation (CLIP); iCLIP