The splicing factor U2AF(35) mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing

The splicing factor U2AF (U2 snRNP auxiliary factor) is a heterodimer with subunits of 65 and 35 kD (U2AF(65) and U2AF(35)). U2AF(65) binds specifically to 3' splice sites, but previous studies failed to demonstrate a function for U2AF(35). Here, we report that U2AF(35) is required for constitutive splicing and also functions as a mediator of enhancer-dependent splicing. Nuclear extracts deficient in U2AF(35) were inactive; however, both constitutive and enhancer-dependent splicing could be restored by the addition of purified recombinant U2AF(35). In vitro protein-RNA interaction studies with pre-mRNAs containing either a constitutive or regulated splicing enhancer revealed that U2AF(35) directly mediates interactions between U2AF(65) and proteins bound to the enhancers. Thus, U2AF(35) functions as a bridge between U2AF(65) and the enhancer complex to recruit U2AF(65) to the adjacent intron.