Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65)

U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF(65) and U2AF(35). U2AF(65) binds to the polypyrimidine (Py) tract upstream from the 3' splice site an d plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF(35) facilitates U2AF(65) binding through a network of protein-pro tein interactions with other splicing factors, but the requirement and func tion of U2AF(35) remain controversial. Here we show that recombinant U2AF(6 5) is sufficient to activate the splicing of two constitutively spliced pre -mRNAs in extracts that were chromatographically depleted of U2AF. In contr ast, U2AF(65), U2AF(35), and the interaction between them are required for splicing of an immunoglobulin mu pre-RNA containing an intron with a weak P y tract and a purine-rich exonic splicing enhancer. Remarkably, splicing ac tivation by U2AF(35) occurs without changes in U2AF(65) cross-linking to th e Py tract. These results reveal substrate-specific requirements for U2AF(3 5) and a novel function for this factor in pre-mRNA splicing.