The on/off state of the binary switch gene Sex-lethal (Sxl), which controls somatic sexual development in Drosophila melanogaster, is regulated at the level of alternative splicing. In males, in which the gene is off, the default splicing machinery produces nonfunctional mRNAs; in females, in which the gene is on, the autoregulatory activity of the Sxl proteins directs the splicing machinery to produce functional mRNAs. We have used germ line transformation to analyze the mechanism of default and regulated splicing. Our results demonstrate that a blockage mechanism is employed in Sxl autoregulation. However, in contrast to transformer, in which Sxl appears to function by preventing the interaction of splicing factors with the default 3' splice site, a different strategy is used in autoregulation. (i) Multiple cis-acting elements, both upstream and downstream of the male exon, are required. (ii) These cis-acting elements are distant from the splice sites they regulate, suggesting that the Sxl protein cannot function in autoregulation by directly competing with splicing factors for interaction with the regulated splice sites. (iii) The 5' splice site of the male exon appears to be dominant in regulation while the 3' splice site plays a subordinate role.Posttranscriptional regulatory mechanisms play a key role in cell fate decisions in many developmental pathways. In the somatic sexual-development pathway of the fruit fly, Drosophila melanogaster, both the maintenance and elaboration of pathway choice are controlled at the level of alternative pre-mRNA splicing (2, 21). The binary switch gene Sex-lethal (Sxl) sits at the top of the somatic sexualdevelopment pathway and functions in both determination and differentiation. The activity state of the Sxl gene is chosen during early embryogenesis in response to the primary sex determination signal, the ratio of X chromosomes to autosomes (9,14,27,47). Sxl is turned on in females, while it remains off in males. Activation in females triggers an autoregulatory feedback loop in which female Sxl proteins promote their own synthesis by directing the femalespecific splicing of Sxl primary transcripts (3,4 regulates the splicing of transformer (tra) RNA (5). When Sxl is on, it directs the female-specific splicing of tra primary transcripts, producing mRNAs which have an intact open reading frame. The resulting tra protein, together with the constitutively expressed tra-2 protein, then activates the female-specific splice site of doublesex (dsx) to generate female dsx RNA (7,18,24,43). When Sxl is off, the default splicing of tra gives mRNAs with a truncated open reading frame, and in the absence of functional tra protein, dsx is spliced in the male mode.The mechanism for Sxl regulation of tra splicing is now reasonably well understood. As diagrammed in Fig. 1
MATERIALS AND METHODSPlasmid construction and Drosophila transformation. TheSxl miniconstruct diagrammed in Fig. 2 was made by first isolating a 310-bp PstI-HpaII fragment of genomic Sxl which contains exon 4 and upstrea...