title: Comprehensive Identification of RNA-Binding Proteins by RNA Interactome Capture.
abstract: RNA associates with RNA-binding proteins (RBPs) from synthesis to decay, forming dynamic ribonucleoproteins (RNPs). In spite of the preeminent role of RBPs regulating RNA fate, the scope of cellular RBPs has remained largely unknown. We have recently developed a novel and comprehensive method to identify the repertoire of active RBPs of cultured cells, called RNA interactome capture. Using in vivo UV cross-linking on cultured cells, proteins are covalently bound to RNA if the contact between the two is direct ("zero distance"). Protein-RNA complexes are purified by poly(A) tail-dependent oligo(dT) capture and analyzed by quantitative mass spectrometry. Because UV irradiation is applied to living cells and purification is performed using highly stringent washes, RNA interactome capture identifies physiologic and direct protein-RNA interactions. Applied to HeLa cells, this protocol revealed the near-complete repertoire of RBPs, including hundreds of novel RNA binders. Apart from its RBP discovery capacity, quantitative and comparative RNA interactome capture can also be used to study the responses of the RBP repertoire to different physiological cues and processes, including metabolic stress, differentiation, development, or the response to drugs.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK. alfredo.castellopalomares@bioch.ox.ac.uk.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Castello
authors.affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
authors.forename: Rastislav
authors.initials: R
authors.lastname: Horos
authors.affiliation: German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Strein
authors.affiliation: German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Fischer
authors.affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Eichelbaum
authors.affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany.|Genome Biology Unit, EMBL Heidelberg, Heidelberg, Germany.|Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Lars M
authors.initials: LM
authors.lastname: Steinmetz
authors.affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Krijgsveld
authors.affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
authors.forename: Matthias W
authors.initials: MW
authors.lastname: Hentze
mesh_terms: D006367:HeLa Cells; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D046912:Multiprotein Complexes / Q000235:genetics / Q000302:isolation & purification*; D040901:Proteomics; D012313:RNA / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D012261:Ribonucleoproteins / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D046912:Multiprotein Complexes; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D012313:RNA
keywords: Gene expression; Posttranscriptional regulation; Proteome; Proteomics; RNA; RNA interactome capture; RNA-binding protein