FUNCTIONAL-ANALYSIS OF PREMESSENGER RNA SPLICING FACTOR SF2 ASF STRUCTURAL DOMAINS/

Human pre-mRNA splicing factor SF2/ASF has an activity required for ge neral splicing in vitro and promotes utilization of proximal alternati ve 5' splice sites in a concentration-dependent manner by opposing hnR NP A1. We introduced selected mutations in the N-terminal RNA recognit ion motif (RRM and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and altern ative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site s election, demonstrating that these activities involve distinct molecul ar interactions. Specific protein - RNA contacts mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for cons titutive splicing, although they are not required for RRM-mediated bin ding to pre-mRNA. The RS domain is also required for constitutive spli cing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM a nd a central degenerate RRM repeat in binding to RNA. These two domain s are sufficient for alternative splicing activity in the absence of a n RS domain.