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

Introns are defined by sequences that bind components of the splicing machinery. The branchpoint consensus, polypyrimidine (poly(Y)) tract, and AG at the splice boundary comprise the mammalian 3′ splice site1. 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 responsible for AG recognition has never been identified. A key player in 3′ splice site definition is the essential heterodimeric splicing factor U2AF, which facilitates the interaction of the U2 small nuclear ribonucleoprotein particle with the branch point. The U2AF subunit with a relative molecular mass (Mr 65K) of 65,000 (U2AF65) binds to the poly(Y) tract3,4,5,6,7, whereas the role of the 35K subunit (U2AF35)8 has not been clearly defined. It is not required for splicing in vitro4 but it plays a critical role in vivo9,10. Caenorhabiditis elegans introns have a highly conserved U4CAG/R at their 3′ splice sites instead of branch-point and poly(Y) consensus sequences11. Nevertheless, C. elegans has U2AF (refs 10, 12). Here we show that both U2AF subunits crosslink to the 3′ splice site. Our results suggest that the U2AF65–U2AF35 complex identifies the U4CAG/R, with U2AF35 being responsible for recognition of the canonical AG.