Functional recognition of the 3 ' splice site AG by the splicing factor U2AF(35)

In metazoans, spliceosome assembly is initiated through recognition of the 5' splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuc lear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF (refs 1, 2), U2AF is a heterodimer comprising a large subunit, U2AF(65), and a small su bunit, U2AF(35) (ref. 3). U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro(4). In comparison, the role of U2AF3 5 has been puzzling: U2AF(35) is highly conserved(5-7) and is required for viabilit(6,7), but can be dispensed with for splicing in vitro(4,8,9). Here we use sire-specific crosslinking to show that very early during spliceoso me assembly U2AF directly contacts the 3' splice site. Mutational analysis and in vitro genetic selection indicate that U2AF35 has a sequence-specific RNA-binding activity that recognizes the 3'-splice-site consensus, AG/G. W e show that for introns with weak polypyrimidine tracts, the U2AF(35)-3'-sp lice-site interaction is critical for U2AF binding and splicing. Our result s demonstrate a new biochemical activity of U2AF(35) identify the factor th at initially recognizes the 3' splice site, and explain why the AG dinucleo tide is required for the first step of splicing for some but not all intron s.