Switch in 3 ' splice site recognition between exon definition and splicingcatalysis is important for Sex-lethal autoregulation

Maintenance of female sexual identity in Drosophila melanogaster involves a n autoregulatory loop in which the protein Sex-lethal (SXL) promotes skippi ng of exon 3 from its ann pre-mRNA. me have used transient transfection of Drosophila Schneider cells to analyze the role of exon 3 splice sites in re gulation. Our results indicate that exon 3 repression requires competition between the 5 ' splice sites of exons 2 and 3 but is independent of their r elative strength. Two 3 ' splice site AG's precede exon 3. We report here t hat, while the distal site plays a critical role in defining the exon, the proximal site is preferentially used for the actual splicing reaction, argu ing for a switch in 3 ' splice site recognition between exon definition and splicing catalysis. Remarkably, the presence of the two 3 ' splice sites i s important for the efficient regulation by SXL, suggesting that SXL interf eres with molecular events occurring between initial splice site communicat ion across the exon and the spice site pairing that leads to intron removal .