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′ splice site (SS), and both factors affect 5′ 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′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.
Highlights
SRSF1 is one of the most-studied regulators of alternative splicing
We suggest that the recruitment of SRSF1 to a U1 snRNP at a 50 splice sites (50SSs) 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
We have shown previously that the activity of enhancer sequences (ESEs) is limited by their occupancy, consistent with transient binding by SRSF1 and stabilization of one molecule by direct interactions based on diffusional encounters with splice site-associated factors, since their effects are not blocked by the incorporation of intervening nonRNA linkers (Jobbins et al, 2018)
Summary
SRSF1 is one of the most-studied regulators of alternative splicing. It is the archetypal member of the family of SR proteins, proteins that have one or two RNA recognition motif (RRM)-type RNA-binding domains and a C-terminal RS domain rich in arginine-serine dipeptides that can be phosphorylated extensively. The resultant model for activation of splicing by SRSF1 is that it binds exonic splicing enhancer sequences (ESEs) (Graveley & Maniatis, 1998; Sanford et al, 2009; Clery et al, 2013; Pandit et al, 2013; Ray et al, 2013; Anczukow et al, 2015) and recruits limiting splicing factors such as U1 snRNPs or U2-associated proteins to 50 or 30 splice sites, respectively, by direct protein–protein interactions that stabilize the association of the splicing factor with the pre-mRNA (Eperon et al, 1993; Lavigueur et al, 1993; Wu & Maniatis, 1993; Amrein et al, 1994; Kohtz et al, 1994; Staknis & Reed, 1994; Jamison et al, 1995; Tarn & Steitz, 1995; Wang et al, 1995; Cao & Garcia-Blanco, 1998; Graveley et al, 2001; Martins de Araujo et al, 2009; Cho et al, 2011; Smith et al, 2014; Akerman et al, 2015).
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