RNA recognition by Npl3p reveals U2 snRNA-binding compatible with a chaperone role during splicing

Nat Commun. 2023 Nov 7;14(1):7166. doi: 10.1038/s41467-023-42962-4.

Abstract

The conserved SR-like protein Npl3 promotes splicing of diverse pre-mRNAs. However, the RNA sequence(s) recognized by the RNA Recognition Motifs (RRM1 & RRM2) of Npl3 during the splicing reaction remain elusive. Here, we developed a split-iCRAC approach in yeast to uncover the consensus sequence bound to each RRM. High-resolution NMR structures show that RRM2 recognizes a 5´-GNGG-3´ motif leading to an unusual mille-feuille topology. These structures also reveal how RRM1 preferentially interacts with a CC-dinucleotide upstream of this motif, and how the inter-RRM linker and the region C-terminal to RRM2 contribute to cooperative RNA-binding. Structure-guided functional studies show that Npl3 genetically interacts with U2 snRNP specific factors and we provide evidence that Npl3 melts U2 snRNA stem-loop I, a prerequisite for U2/U6 duplex formation within the catalytic center of the Bact spliceosomal complex. Thus, our findings suggest an unanticipated RNA chaperoning role for Npl3 during spliceosome active site formation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Nucleic Acid Conformation
  • RNA Splicing*
  • RNA* / metabolism
  • RNA, Small Nuclear / metabolism
  • Ribonucleoprotein, U2 Small Nuclear / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Spliceosomes / metabolism

Substances

  • Ribonucleoprotein, U2 Small Nuclear
  • RNA
  • RNA, Small Nuclear
  • U2 small nuclear RNA
  • NPL3 protein, S cerevisiae