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.