title: Structure, Dynamics, and Interaction of p54(nrb)/NonO RRM1 with 5' Splice Site RNA Sequence.
abstract: p54(nrb)/NonO is a nuclear RNA-binding protein involved in many cellular events such as pre-mRNA processing, transcription, and nuclear retention of hyper-edited RNAs. In particular, it participates in the splicing process by directly binding the 5' splice site of pre-mRNAs. The protein also concentrates in a nuclear body called paraspeckle by binding a G-rich segment of the ncRNA NEAT1. The N-terminal section of p54(nrb)/NonO contains tandem RNA recognition motifs (RRMs) preceded by an HQ-rich region including a threonine residue (Thr15) whose phosphorylation inhibits its RNA binding ability, except for G-rich RNAs. In this work, our goal was to understand the rules that characterize the binding of the p54(nrb)/NonO RRMs to their RNA target. We have done in vitro RNA binding experiments which revealed that only the first RRM of p54(nrb)/NonO binds to the 5' splice site RNA. We have then determined the structure of the p54(nrb)/NonO RRM1 by liquid-state NMR which revealed the presence of a canonical fold (β1α1β2β3α2β4) and the conservation of aromatic amino acids at the protein surface. We also investigated the dynamics of this domain by NMR. The p54(nrb)/NonO RRM1 displays some motional properties that are typical of a well-folded protein with some regions exhibiting more flexibility (loops and β-strands). Furthermore, we determined the affinity of p54(nrb)/NonO RRM1 interaction to the 5' splice site RNA by NMR and fluorescence quenching and mapped its binding interface by NMR, concluding in a classical nucleic acid interaction. This study provides an improved understanding of the molecular basis (structure and dynamics) that governs the binding of the p54(nrb)/NonO RRM1 to one of its target RNAs.
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Duvignaud
authors.forename: Mikaël
authors.initials: M
authors.lastname: Bédard
authors.affiliation: RIKEN Center for Life Science Technologies , Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nagata
authors.affiliation: RIKEN Center for Life Science Technologies , Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.|Faculty of Pharmacy and Research Institute of Pharmaceutical Science, Musashino University , Nishitokyo-shi, Tokyo 202-8585, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Muto
authors.affiliation: RIKEN Systems and Structural Biology Center , 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.|RIKEN Structural Biology Laboratory , 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Stéphane M
authors.initials: SM
authors.lastname: Gagné
authors.forename: Michel
authors.initials: M
authors.lastname: Vincent
mesh_terms: D000818:Animals; D051379:Mice; D034521:Nuclear Matrix-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D012322:RNA Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing; D062085:RNA, Long Noncoding / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012262:Ribonucleoside Diphosphate Reductase; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins
chemical_list: D034521:Nuclear Matrix-Associated Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D062085:RNA, Long Noncoding; D016601:RNA-Binding Proteins; D012262:Ribonucleoside Diphosphate Reductase; D012264:Ribonucleotide Reductases; C577275:NEAT1 long non-coding RNA, mouse; C498690:Rrm1 protein, mouse; C500331:Nono protein, mouse; D004268:DNA-Binding Proteins