title: Protein-RNA networks revealed through covalent RNA marks.
abstract: Protein-RNA networks are ubiquitous and central in biological control. We present an approach termed RNA Tagging that enables the user to identify protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or cross-linking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA via high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The results showed that although RNA-binding proteins productively bind specific RNAs to control their function, they also 'sample' RNAs without exerting a regulatory effect. We used the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. RNA Tagging is well suited to detect and analyze protein-RNA networks in vivo.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Lapointe
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wilinski
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Harriet A J
authors.initials: HA
authors.lastname: Saunders
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Marvin
authors.initials: M
authors.lastname: Wickens
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D001665:Binding Sites; D011485:Protein Binding; D012331:RNA, Fungal / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; C089100:BFR1 protein, S cerevisiae; C510155:PUF3 protein, S cerevisiae; D012331:RNA, Fungal; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins