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 B^act spliceosomal complex. Thus, our findings suggest an unanticipated RNA chaperoning role for Npl3 during spliceosome active site formation.

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Journal Article | Research Support, Non-U.S. Gov't

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Moursy A, Cléry A, Gerhardy S, Betz KM, Rao S, Mazur J, Campagne S, Beusch I, Duszczyk MM, Robinson MD, ... Show all

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