Structure, Dynamics, and Interaction of p54nrb/NonO RRM1 with 5′ Splice Site RNA Sequence

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• 作者：Jean-Baptiste Duvignaud ; Mikaël Bédard ; Takashi Nagata ; Yutaka Muto ; Shigeyuki Yokoyama ; Stéphane M. Gagné ; Michel Vincent
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：May 10, 2016
• 年：2016
• 卷：55
• 期：18
• 页码：2553-2566
• 全文大小：720K
• 年卷期：0
• ISSN：1520-4995

文摘

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.