title: Profiling the Binding Sites of RNA-Binding Proteins with Nucleotide Resolution Using iCLIP.
abstract: The importance of posttranscriptional regulation in cellular metabolism has recently gone beyond what was previously appreciated. The regulatory mechanisms are controlled by RNA-binding proteins (RBPs), which form complexes with RNA and regulate RNA processing, stability, and localization, among others. Consistently, mutations in RBPs result in defects in developmental processes, diseases, and cancer. Gaining deeper insights into the biology of RNA-RBP interactions will lead to a better understanding of regulatory processes and disease development. Several techniques have been developed to capture the properties of RNA-RBP interactions. Furthermore, the development of high-throughput sequencing has broadened the capability of these methods. Here, we summarize individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), a powerful technique that provides genome-wide information on RNA-RBP interactions at nucleotide resolution. In this chapter, we outline the iCLIP protocol and list possible controls that allow a targeted and cost-minimizing optimization of the protocol for an RBP-of-interest. Moreover, we provide notes on experimental design and a troubleshooting guideline for common problems that can occur during iCLIP library preparation.
authors.affiliation: Institute of Molecular Biology gGmbH, Ackermannweg 4, 55128, Mainz, Germany.
authors.forename: F X Reymond
authors.initials: FX
authors.lastname: Sutandy
authors.affiliation: Institute of Molecular Biology gGmbH, Ackermannweg 4, 55128, Mainz, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Hildebrandt
authors.affiliation: Institute of Molecular Biology gGmbH, Ackermannweg 4, 55128, Mainz, Germany. j.koenig@imb-mainz.de.
authors.forename: Julian
authors.initials: J
authors.lastname: König
mesh_terms: D001665:Binding Sites / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009711:Nucleotides / Q000235:genetics; D012313:RNA / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D009711:Nucleotides; D016601:RNA-Binding Proteins; D012313:RNA
keywords: Protein–RNA interactions; RBP binding site; RNA; RNA-binding protein; iCLIP