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


Moursy, Ahmed; Cléry, Antoine; Gerhardy, Stefan; Betz, Katharina M; Rao, Sanjana; Mazur, Jarosław; Campagne, Sébastien; Beusch, Irene; Duszczyk, Malgorzata M; Robinson, Mark D; Panse, Vikram Govind; Allain, Frédéric H-T (2023). RNA recognition by Npl3p reveals U2 snRNA-binding compatible with a chaperone role during splicing. Nature Communications, 14(1):7166.

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

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

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Medical Microbiology
05 Vetsuisse Faculty > Veterinärwissenschaftliches Institut > Institute of Food Safety and Hygiene
07 Faculty of Science > Department of Chemistry
07 Faculty of Science > Institute of Molecular Life Sciences
Dewey Decimal Classification:610 Medicine & health
540 Chemistry
570 Life sciences; biology
Scopus Subject Areas:Physical Sciences > General Chemistry
Life Sciences > General Biochemistry, Genetics and Molecular Biology
Physical Sciences > General Physics and Astronomy
Language:English
Date:7 November 2023
Deposited On:27 Dec 2023 11:10
Last Modified:29 Jun 2024 01:41
Publisher:Nature Publishing Group
ISSN:2041-1723
OA Status:Gold
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1038/s41467-023-42962-4
PubMed ID:37935663
Other Identification Number:PMCID: PMC10630445
  • Content: Published Version
  • Language: English
  • Licence: Creative Commons: Attribution 4.0 International (CC BY 4.0)