Exon‐independent recruitment of SRSF1 is mediated by U1 snRNP stem‐loop 3

Abstract

SRSF1 protein and U1 snRNPs are closely connected splicing factors. They both stimulate exon inclusion, SRSF1 by binding to exonic splicing enhancer sequences (ESEs) and U1 snRNPs by binding to the downstream 5 0 splice site (SS), and both factors affect 5 0 SS selection. The binding of U1 snRNPs initiates spliceosome assembly, but SR proteins such as SRSF1 can in some cases substitute for it. The mechanistic basis of this relationship is poorly understood. We show here by single-molecule methods that a single molecule of SRSF1 can be recruited by a U1 snRNP. This reaction is independent of exon sequences and separate from the U1-independent process of binding to an ESE. Structural analysis and cross-linking data show that SRSF1 contacts U1 snRNA stem-loop 3, which is required for splicing. We suggest that the recruitment of SRSF1 to a U1 snRNP at a 5 0 SS is the basis for exon definition by U1 snRNP and might be one of the principal functions of U1 snRNPs in the core reactions of splicing in mammals.

Exonic splicing code and protein binding sites for calcium

“…ESEs containing GA dinucleotides may bind a subset of SR proteins ( Supplementary Table S2 ) but individual GAY-to-CAC mutations and ESE-to-ESS remodeling of the DADLD site were insufficient to alter exon 4b inclusion levels by SR protein regulators (Figure 5H ). The failure to respond to mutations is consistent with a significant ESE-independent component of SRSF1 action, as proposed ( 124 , 125 ). In the most recent model ( 125 ), SRSF1 could be directly recruited to 5′ss through interactions with stem–loop 3 of U1 small nuclear ribonucleoprotein (snRNP), namely, between SRSF1 RRM1 and the CA motif at the 5′ part of the stem-loop, and between RRM2 and a double-stranded GGA motif, with ESEs showing only low transient SRSF1 occupancy ( 125 , 126 ).…”

“…In the most recent model ( 125 ), SRSF1 could be directly recruited to 5′ss through interactions with stem–loop 3 of U1 small nuclear ribonucleoprotein (snRNP), namely, between SRSF1 RRM1 and the CA motif at the 5′ part of the stem-loop, and between RRM2 and a double-stranded GGA motif, with ESEs showing only low transient SRSF1 occupancy ( 125 , 126 ). In other words, the ESE-independent SRSF1 interactions appearred to be sufficient for connecting U1 and U2 snRNPs ( 125 ).…”

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

“…6). We found that SRSF1 (Jobbins, Campagne et al, 2022) and FUS (Jutzi, Campagne et al, 2020) could interact directly with the SL3 of the human U1 snRNA. The specific interaction between Npl3 and the U2 snRNA reported here implicates a broader role for the U snRNAs.…”

“…6). We found that SRSF1 (Jobbins, Campagne et al, 2022) and FUS (Jutzi, Campagne et al, 2020) could interact directly with the SL3 of the human U1 snRNA.…”

Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly

“…U1 snRNP-mediated SR protein displacement appears to involve a complex and dynamic mechanism. SRSF1 interacts with U1-70k (37) and the stem-loop III of U1 snRNA (43).…”