Molecular genetic analysis of the heterodimeric splicing factor U2AF: the RS domain on either the large or small Drosophila subunit is dispensable in vivo.
The pre-mRNA splicing factor U2AF (U2 snRNP auxiliary factor) has an essential role in 3' splice site selection. U2AF binds the intron pyrimidine tract between the branchpoint and the 3' splice site and recruits U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. Both subunits contain a domain enriched in arginine-serine dipeptide repeats termed an RS domain. The two U2AF RS domains have been assigned essential and independent roles in spliceosome assembly in vitro-the hU2AF65 RS domain is required to target U2 snRNP to the branch site and the hU2AF35 RS domain is necessary for protein-protein interactions with constitutive and alternative splicing factors. We have investigated the functional requirements for the RS domains on the Drosophila U2AF homolog in vivo. In sharp contrast to its essential role in U2 snRNP recruitment in vitro, the RS domain on the Drosophila large subunit homolog (dU2AF50) was completely dispensable in vivo. Prompted by this unexpected result, we analyzed the RS domain on the Drosophila small subunit homolog (dU2AF38). Despite its requirement for enhancer-dependent splicing activity in vitro, the dU2AF38 RS domain was also inessential in vivo. Finally, we have tested whether the Drosophila U2AF heterodimer requires any RS domain. Flies mutant for both the small and large subunits could not be rescued by dU2AF50deltaRS and dU2AF38deltaRS transgenes. Therefore, in contrast to the separate roles assigned to the U2AF RS domains in vitro, our genetic data suggest that they may have redundant functions in vivo.
Rudner
D Z
DZ
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3204 USA.
Breger
K S
KS
Rio
D C
DC
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D004330:Drosophila / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000235:genetics* / Q000378:metabolism; D000072668:Splicing Factor U2AF
D001426:Bacterial Proteins; D009687:Nuclear Proteins; D012322:RNA Precursors; D012261:Ribonucleoproteins; C063999:SR protein, bacteria; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human