SEQUENCE-SPECIFIC RNA-BINDING BY AN SR PROTEIN REQUIRES RS DOMAIN PHOSPHORYLATION - CREATION OF AN SRP40-SPECIFIC SPLICING ENHANCER

We showed previously that ASF/SF2, a member of the SR protein family o f splicing factors, can activate a splicing enhancer element composed of high-affinity ASF/SF2 binding sites, To determine whether other SR proteins can behave similarly, we selected a high-affinity RNA-binding site (B1) for the SR protein SRp40. Strikingly, the success of this s election was completely dependent on phosphorylation of the RS domain, as unphosphorylated SRp40 failed to select. specific sequences, We sh ow (hat three copies of B1 function as a strong splicing enhancer, act ivating an intron with suboptimal splicing signals in nuclear extracts , Enhancer activity in S100 extracts (which lack SR proteins) required SRp40 and a nuclear fraction previously Found to be required for ASF/ SF2-dependent splicing. Importantly, enhancer activity was lost when S Rp40 was replaced by ASF/SF2 or SC35, and SRp40 was the only classical SR protein found to be associated with the enhancer, Together, our re sults indicate that phosphorylation-dependent, sequence-specific RNA b inding can impart unique activities to individual SR proteins.