Abstract
Serine/arginine-rich (SR) proteins are essential splicing factors with one or two RNA-recognition motifs (RRMs) and a C-terminal arginine- and serine-rich (RS) domain. SR proteins bind to exonic splicing enhancers via their RRM(s), and from this position are thought to promote splicing by antagonizing splicing silencers, recruiting other components of the splicing machinery through RS-RS domain interactions, and/or promoting RNA base-pairing through their RS domains. An RS domain tethered at an exonic splicing enhancer can function as a splicing activator, and RS domains play prominent roles in current models of SR protein functions. However, we previously reported that the RS domain of the SR protein SF2/ASF is dispensable for in vitro splicing of some pre-mRNAs. We have now extended these findings via the identification of a short inhibitory domain at the SF2/ASF N-terminus; deletion of this segment permits splicing in the absence of this SR protein's RS domain of an IgM pre-mRNA substrate previously classified as RS-domain-dependent. Deletion of the N-terminal inhibitory domain increases the splicing activity of SF2/ASF lacking its RS domain, and enhances its ability to bind pre-mRNA. Splicing of the IgM pre-mRNA in S100 complementation with SF2/ASF lacking its RS domain still requires an exonic splicing enhancer, suggesting that an SR protein RS domain is not always required for ESE-dependent splicing activation. Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing.
Highlights
The SR proteins are a family of conserved splicing factors that consist of either one or two N-terminal RNA recognition motifs (RRM) and a C-terminal arginine- and serine-rich (RS) domain [1,2]
SR protein RS domains were thought to be essential for constitutive splicing, because a recombinant SR protein lacking its RS domain was unable to complement S100 for splicing of constitutive substrates
What these early experiments may have been demonstrating was the importance of sequences preceding RRM1 in influencing the ability of SR proteins to be recruited to pre-mRNA
Summary
The SR proteins are a family of conserved splicing factors that consist of either one or two N-terminal RNA recognition motifs (RRM) and a C-terminal arginine- and serine-rich (RS) domain [1,2]. SR proteins bind to ESEs via their RRM(s) [10], whereas their RS domains are thought to function as protein-protein interaction modules that facilitate exon inclusion by recruiting components of the basal splicing machinery to the flanking 59 and 39 splice sites early in splice-site recognition [11]. It was proposed that SR proteins can promote recruitment of the U1 snRNP to the 59 splice site through SR protein RS-domain-mediated interactions with U1-70K [14]. Enhancer-bound SR proteins are thought to escort U2AF65 to the 39 splice site polypyrimidine tract through RS-domain-mediated recruitment of U2AF35 [16,17,18]. The aforementioned functions of SR proteins are assumed to occur via RS-domainmediated protein-protein interactions, it has not yet been demonstrated that such interactions occur in the context of a functional spliceosome [24]
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