Both subunits of U2AF recognize the 3 ' splice site in Caenorhabditis elegans

Introns are defined by sequences that bind components of the splicing machi nery. The branchpoint consensus, polypyrimidine (poly(Y)) tract, and AG at the splice boundary comprise the mammalian 3' splice site(1), Although the AG is crucial for the recognition of introns with relatively short poly(Y) tracts, which are termed 'AG-dependent introns'(2), the molecule responsibl e for AG recognition has never been identified. A key player in 3' splice s ite definition is the essential heterodimeric splicing factor U2AF, which f acilitates the interaction of the U2 small nuclear ribonucleoprotein partic le with the branch point. The U2AF subunit with a relative molecular mass ( M-r 65K) of 65,000 (U2AF(65)) binds to the poly(Y) tract(3-7), whereas the role of the 35K subunit (U2AF(35))(8) has not been clearly defined. It is n ot required for splicing in vitro(4) but it plays a critical role in vivo(9 ,10) Caenorhabiditis elegans introns have a highly conserved U(4)CAG/R at t heir 3' splice sites instead of branch-point and poly(Y) consensus sequence s(11). Nevertheless, C. elegans has U2AF (refs 10, 12). Here we show that b oth U2AF subunits crosslink to the 3' splice site. Our results suggest that the U2AF(65)-U2AF(35) complex identifies the U(4)CAG/R, with U2AF35 being responsible for recognition of the canonical AG.