title: RNAcompete methodology and application to determine sequence preferences of unconventional RNA-binding proteins.
abstract: RNA-binding proteins (RBPs) participate in diverse cellular processes and have important roles in human development and disease. The human genome, and that of many other eukaryotes, encodes hundreds of RBPs that contain canonical sequence-specific RNA-binding domains (RBDs) as well as numerous other unconventional RNA binding proteins (ucRBPs). ucRBPs physically associate with RNA but lack common RBDs. The degree to which these proteins bind RNA, in a sequence specific manner, is unknown. Here, we provide a detailed description of both the laboratory and data processing methods for RNAcompete, a method we have previously used to analyze the RNA binding preferences of hundreds of RBD-containing RBPs, from diverse eukaryotes. We also determine the RNA-binding preferences for two human ucRBPs, NUDT21 and CNBP, and use this analysis to exemplify the RNAcompete pipeline. The results of our RNAcompete experiments are consistent with independent RNA-binding data for these proteins and demonstrate the utility of RNAcompete for analyzing the growing repertoire of ucRBPs.
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Debashish
authors.initials: D
authors.lastname: Ray
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Kevin C H
authors.initials: KCH
authors.lastname: Ha
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Kate
authors.initials: K
authors.lastname: Nie
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Hong
authors.initials: H
authors.lastname: Zheng
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Electronic address: t.hughes@utoronto.ca.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Hughes
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Department of Computer Science, University of Toronto, Toronto, Ontario M5S 3G4, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Electronic address: quaid.morris@utoronto.ca.
authors.forename: Quaid D
authors.initials: QD
authors.lastname: Morris
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D039223:Cleavage And Polyadenylation Specificity Factor / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D011485:Protein Binding; D000072417:Protein Domains; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: C433227:CNBP protein, human; D039223:Cleavage And Polyadenylation Specificity Factor; D017931:DNA Primers; C580829:Nudt21 protein, human; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012313:RNA
keywords: Binding site; CNBP; DNA microarray; NUDT21; RNA-binding protein; RNAcompete