Functional recognition of the 3' splice site AG by the splicing factor U2AF35.
In metazoans, spliceosome assembly is initiated through recognition of the 5' splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF. U2AF is a heterodimer comprising a large subunit, U2AF65, and a small subunit, U2AF35. U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro. In comparison, the role of U2AF35 has been puzzling: U2AF35 is highly conserved and is required for viability, but can be dispensed with for splicing in vitro. Here we use site-specific crosslinking to show that very early during spliceosome assembly U2AF35 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. We show that for introns with weak polypyrimidine tracts, the U2AF35-3'-splice-site interaction is critical for U2AF binding and splicing. Our results demonstrate a new biochemical activity of U2AF35, identify the factor that initially recognizes the 3' splice site, and explain why the AG dinucleotide is required for the first step of splicing for some but not all introns.
Wu
S
S
Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605, USA.
Romfo
C M
CM
Nilsen
T W
TW
Green
M R
MR
D000227:Adenine Nucleotides / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D006150:Guanine Nucleotides / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins; D011485:Protein Binding; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D012326:RNA Splicing; D012261:Ribonucleoproteins / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism; D000072668:Splicing Factor U2AF
D000227:Adenine Nucleotides; D006150:Guanine Nucleotides; D009687:Nuclear Proteins; D012322:RNA Precursors; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C076753:U2AF1 protein, human