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Structure, Dynamics, and Interaction of p54 nrb /NonO RRM1 with 5′ Splice Site RNA Sequence

2016; American Chemical Society; Volume: 55; Issue: 18 Linguagem: Inglês

10.1021/acs.biochem.5b01240

1943-295X

Jean‐Baptiste Duvignaud, Mikaël Bédard, Takashi Nagata, Y. Muto, Shigeyuki Yokoyama, Stéphane M. Gagné, Michel Vincent,

RNA modifications and cancer

p54nrb/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 p54nrb/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 p54nrb/NonO RRMs to their RNA target. We have done in vitro RNA binding experiments which revealed that only the first RRM of p54nrb/NonO binds to the 5′ splice site RNA. We have then determined the structure of the p54nrb/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 p54nrb/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 p54nrb/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 p54nrb/NonO RRM1 to one of its target RNAs.