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The human splicing factor U2 auxiliary factor (hsU2AF) is comprised of two interacting subunits of 65 and 35 kDa. Previously we identified the Schizosaccharomyces pombe homolog, spU2AF59, of the human large subunit. We have screened a fission yeast cDNA library in search of proteins that interact with spU2AF59 using the yeast two-hybrid system and have identified a homolog of the hsU2AF35 subunit. The S. pombe U2AF small subunit is a single copy gene that encodes a protein which shares 55% amino acid identity and 17% similarity with the human small subunit. Unlike the human protein, the yeast protein lacks an arginine/serine-rich region. The predicted molecular mass of the spU2AF small subunit is 23 kDa. The region of spU2AF59 that interacts with spU2AF23 is similar to the region in which the human small and large subunits interact.