title: Identification of miRNA-Target RNA Interactions Using CLASH.
abstract: We present a detailed protocol for the experimental identification of miRNA-target RNA interaction sites using cross-linking, ligation, and sequencing of hybrids (CLASH). The basis of the technique is the purification of UV-stabilized Argonaute (AGO)-RNA complexes assembled in living cells, with subsequent ligation of AGO-associated RNA-RNA duplexes to form chimeric RNAs. Following cDNA synthesis, DNA library preparation and high-throughput sequencing, interacting RNA molecules are unambiguously identified as chimeric reads in bioinformatic analysis of sequencing data. CLASH potentially recovers any RNA duplex that is bound by RNA-binding protein, so modified approaches would be suitable for the identification of many other inter- and intramolecular RNA-RNA interactions. Since CLASH analysis is independent of bioinformatic predictions it allows the identification and analysis of RNA targeting rules in an unbiased way.
authors.affiliation: Wellcome Trust Centre for Cell Biology, The University of Edinburgh, King's Buildings, Edinburgh, EH9 3BF, UK. olahelwak@yahoo.com.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Helwak
authors.affiliation: Wellcome Trust Centre for Cell Biology, The University of Edinburgh, King's Buildings, Edinburgh, EH9 3BF, UK. d.tollervey@ed.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Tollervey
mesh_terms: D060565:Argonaute Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D019295:Computational Biology / Q000379:methods*; D005786:Gene Expression Regulation; D059014:High-Throughput Nucleotide Sequencing; D047468:Immunoprecipitation / Q000379:methods*; D035683:MicroRNAs / Q000235:genetics / Q000302:isolation & purification*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D060565:Argonaute Proteins; D035683:MicroRNAs; D016601:RNA-Binding Proteins; D012313:RNA
keywords: Argonaute; CLASH; CLIP; Protein-RNA interaction s; RNA cross-linking; RNA-RNA interactions; UV cross-linking; miRNA target identification; microRNA