The structure of yeast Npl3 bound to RNA reveals a cooperative sequence-specific recognition and an RNA chaperone role in splicing

Moursy, A.; Clery, A.; Gerhardy, S.; Betz, K. M.; Rao, S.; Campagne, S.; Beusch, I.; Duszczyk, M. M.; Robinson, M. D.; Panse, V. G.; Allain, F. H.- T.

doi:10.1101/2022.05.24.493251

bioRxiv · 10.1101/2022.05.24.493251

The structure of yeast Npl3 bound to RNA reveals a cooperative sequence-specific recognition and an RNA chaperone role in splicing

Moursy, A.·Clery, A.·Gerhardy, S.·Betz, K. M.·Rao, S.·Campagne, S.·Beusch, I.·Duszczyk, M. M.·Robinson, M. D.·Panse, V. G.·Allain, F. H.- T.

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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 contributes 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 provide insights into an unanticipated RNA chaperoning role for Npl3 during spliceosome active site formation.

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Moursy, A., Clery, A., Gerhardy, S., Betz, K. M., Rao, S., Campagne, S., Beusch, I., Duszczyk, M. M., Robinson, M. D., Panse, V. G., Allain, F. H.- T.. 2022-05-25. The structure of yeast Npl3 bound to RNA reveals a cooperative sequence-specific recognition and an RNA chaperone role in splicing. https://doi.org/10.1101/2022.05.24.493251

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