Evidence for Substrate-Specific Requirement of the Splicing Factor U2AF35 and for Its Function after Polypyrimidine Tract Recognition by U2AF65

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Abstract
U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF65 and U2AF35. U2AF65 binds to the polypyrimidine (Py) tract upstream from the 3′ splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF35 facilitates U2AF65 binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF35 remain controversial. Here we show that recombinant U2AF65 is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF65, U2AF35, and the interaction between them are required for splicing of an immunoglobulin μ pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF35 occurs without changes in U2AF65cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF35 and a novel function for this factor in pre-mRNA splicing.