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Synergistic roles for human U1 snRNA stem-loops in pre-mRNA splicing

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posted on 2021-06-09, 11:00 authored by William Martelly, Bernice Fellows, Paul Kang, Ajay Vashisht, James A. Wohlschlegel, Shalini Sharma

During spliceosome assembly, interactions that bring the 5′ and 3′ ends of an intron in proximity are critical for the production of mature mRNA. Here, we report synergistic roles for the stem-loops 3 (SL3) and 4 (SL4) of the human U1 small nuclear RNA (snRNA) in maintaining the optimal U1 snRNP function, and formation of cross-intron contact with the U2 snRNP. We find that SL3 and SL4 bind distinct spliceosomal proteins and combining a U1 snRNA activity assay with siRNA-mediated knockdown, we demonstrate that SL3 and SL4 act through the RNA helicase UAP56 and the U2 protein SF3A1, respectively. In vitro analysis using UV crosslinking and splicing assays indicated that SL3 likely promotes the SL4-SF3A1 interaction leading to enhancement of A complex formation and pre-mRNA splicing. Overall, these results highlight the vital role of the distinct contacts of SL3 and SL4 in bridging the pre-mRNA bound U1 and U2 snRNPs during the early steps of human spliceosome assembly.

Funding

This work was supported by funds from the National Institutes of General Medical Sciences [GM127464 to SS and GM089778 to JAW], The American Cancer Society [The Institutional Research Grant 74-001-34-IRG to SS], and The Valley Research Partnership Program [P1-4009 to SS and WM]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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