title: Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis.
abstract: The identification of sites where RNA-binding proteins (RNABPs) interact with target RNAs opens the door to understanding the vast complexity of RNA regulation. UV cross-linking and immunoprecipitation (CLIP) is a transformative technology in which RNAs purified from in vivo cross-linked RNA-protein complexes are sequenced to reveal footprints of RNABP:RNA contacts. CLIP combined with high-throughput sequencing (HITS-CLIP) is a generalizable strategy to produce transcriptome-wide maps of RNA binding with higher accuracy and resolution than standard RNA immunoprecipitation (RIP) profiling or purely computational approaches. The application of CLIP to Argonaute proteins has expanded the utility of this approach to mapping binding sites for microRNAs and other small regulatory RNAs. Finally, recent advances in data analysis take advantage of cross-link-induced mutation sites (CIMS) to refine RNA-binding maps to single-nucleotide resolution. Once IP conditions are established, HITS-CLIP takes ∼8 d to prepare RNA for sequencing. Established pipelines for data analysis, including those for CIMS, take 3-4 d.
authors.affiliation: 1] Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, USA. [2].
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Moore
authors.affiliation: 1] Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, USA. [2] [3].
authors.forename: Chaolin
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, USA.
authors.forename: Emily Conn
authors.initials: EC
authors.lastname: Gantman
authors.affiliation: Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, USA.
authors.forename: Aldo
authors.initials: A
authors.lastname: Mele
authors.affiliation: Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, USA.
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Darnell
authors.affiliation: 1] Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, USA. [2].
authors.forename: Robert B
authors.identifier: 0000000251348088
authors.initials: RB
authors.lastname: Darnell
mesh_terms: D060565:Argonaute Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D047468:Immunoprecipitation / Q000379:methods*; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: D060565:Argonaute Proteins; D003432:Cross-Linking Reagents; D016601:RNA-Binding Proteins; D012313:RNA