title: Cross-linking and immunoprecipitation of nuclear RNA-binding proteins.
abstract: The systematic identification of in vivo targets of nuclear RNA-binding proteins (RBPs) is crucial to elucidate the physiological functions of each RBP. However, it has been difficult to distinguish real targets from nonspecifically bound RNAs and to determine the exact binding sites of each RBP by using a conventional RNA-immunoprecipitation (RIP) method. Photoactivatable Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation (PAR-CLIP) is a recently developed method that relies on RNA-protein cross-linking to reduce the contamination of nonspecifically bound RNAs. Furthermore, in combination with high-throughput sequencing followed by bioinformatic analysis, the exact RBP-binding sites can be identified at a single nucleotide resolution. Here, we describe in detail a PAR-CLIP protocol to prepare cDNA libraries for high-throughput sequencing from RNA fragments that are bound to RBPs not only in the nucleus but also in the cytoplasm.
authors.affiliation: Department of RNA Biology and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Quan
authors.initials: Q
authors.lastname: Li
authors.forename: Yuri
authors.initials: Y
authors.lastname: Uemura
authors.forename: Yukio
authors.initials: Y
authors.lastname: Kawahara
mesh_terms: D001665:Binding Sites; D019295:Computational Biology; D003432:Cross-Linking Reagents; D015723:Gene Library; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D009687:Nuclear Proteins / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017423:Sequence Analysis, RNA
chemical_list: D003432:Cross-Linking Reagents; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D012313:RNA