title: Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data.
abstract: Mammalian RNA complexity is regulated through interactions of RNA-binding proteins (RBPs) with their target transcripts. High-throughput sequencing together with UV-crosslinking and immunoprecipitation (HITS-CLIP) is able to globally map RBP-binding footprint regions at a resolution of ~30-60 nucleotides. Here we describe a systematic way to analyze HITS-CLIP data to identify exact crosslink sites, and thereby determine protein-RNA interactions at single-nucleotide resolution. We found that reverse transcriptase used in CLIP frequently skips the crosslinked amino-acid-RNA adduct, resulting in a nucleotide deletion. Genome-wide analysis of these crosslinking-induced mutation sites (CIMS) in HITS-CLIP data for Nova and Argonaute (Ago) proteins in mouse brain tissue revealed deletions in ~8-20% of mRNA tags, which mapped to Nova and Ago binding sites on mRNA or miRNA. CIMS analysis provides a general and more precise means of mapping protein-RNA interactions than currently available methods and insight into the biochemical properties of such interactions in living tissues.
authors.affiliation: Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA. czhang@rockefeller.edu
authors.forename: Chaolin
authors.initials: C
authors.lastname: Zhang
authors.forename: Robert B
authors.initials: RB
authors.lastname: Darnell
mesh_terms: D009711:Nucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D009711:Nucleotides; D011506:Proteins; D012313:RNA