Alternative pre-mRNA splicing is an important mechanism for regulating gene expression in higher eukaryotes. It is estimated that over 70% of all genes are alternatively spliced as a means for producing functionally diverse proteins from a single gene. The changes in mRNA due to alternative splicing can function in a number of developmental processes, including sex-determination and neuronal development. Identifying and understanding the role of cis-acting sequences involved in regulating alternative splicing will help to elucidate the mechanisms that govern alternative splicing and protein diversity. ^ The Drosophila fruitless (fru) gene is a component of the sex-determination cascade and plays central role in determining male sexual behavior. The fru pre-mRNA is alternatively spliced in a sex-specific manner and involves the selection of alternative 5′ splice sites. The upstream male-specific splice site is used by default while the downstream female-specific 5′ splice site is activated by the splicing regulators Transformer (Tra) and Transformer 2 (Tra2). Tra and Tra2 function by binding to three repeats of a 13 nt element located immediately upstream of the female-specific splice site. It has previously been proposed that the splicing enhancer complex functions by recruiting UI snRNP to the female-specific 5′ splice site. However, we find that in the absence of Tra and Tra2 the female-specific 5′ splice site is actively repressed in a manner requiring an intact UI snRNP and the hnRNP protein, PSI. This suggests that the main function of Tra and Tra2 is to activate female-specific splicing by counteracting a repressor rather than by actively recruiting UI snRNP. ^ Through alternative splicing most genes produce transcripts that can generate a relatively modest number of isoforms, however, there are several examples of genes that can potentially generate thousands of alternatively spliced mRNAs. The Drosophila Dscam gene encodes an axon guidance receptor that potentially generates 38,016 different isoforms due to alternative splicing of 95 of its 115 exons. The alternative exons are organized into four clusters that are spliced in a mutually exclusive manner. Here we describe an RNA secondary structural element required for efficient inclusion of all twelve variable exons in the exon 4 cluster, term the inclusion stem (iStem). The iStem resides in the intron between exon 3 and the first exon 4 variant. Interestingly, though the iStem primarily functions as a master regulator that governs inclusion or exclusion of the entire exon 4 cluster, it does not play a significant role in determining which variable exon is selected. Thus, the iStem is a novel type of regulatory element that simultaneously controls the splicing of multiple alternative exons. Taken together, our data suggest a new mechanism for the function of 5′ splice site enhancer-dependent alternative splicing of the Drosophila fru pre-mRNA and we have identified the iStem, a novel type of regulatory element that simultaneously controls whether or not any of several exons are included in the mRNA.
