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pubmed:abstractText |
The Drosophila fruitless (fru) gene encodes a transcription factor that essentially regulates all aspects of male courtship behavior. The use of alternative 5'-splice sites generates fru isoforms that determine gender-appropriate sexual behaviors. Alternative splicing of fru is regulated by TRA and TRA2 and depends on an exonic splicing enhancer (fruRE) consisting of three 13-nucleotide repeat elements, nearly identical to those that regulate alternative sex-specific 3'-splice site choice in the doublesex (dsx) gene. dsx has provided a useful model system to investigate the mechanisms of enhancer-dependent 3'-splice site choice. However, little is known about enhancer-dependent regulation of alternative 5'-splice sites. The mechanisms of this process were investigated using an in vitro system in which recombinant TRA/TRA2 could activate the female-specific 5'-splice site of fru. Mutational analysis demonstrated that one 13-nucleotide repeat element within the fruRE is required and sufficient to activate the regulated female-specific splice site. As was established for dsx, the fruRE can be replaced by a short element encompassing tandem 13-nucleotide repeat elements, by heterologous splicing enhancers, and by artificially tethering a splicing activator to the pre-mRNA. Complementation experiments showed that Ser/Arg-rich proteins facilitate enhancer-dependent 5'-splice site activation. We conclude that splicing enhancers function similarly in activating regulated 5'- and 3'-splice sites. These results suggest that exonic splicing enhancers recruit multiple spliceosomal components required for the initial recognition of 5'- and 3'-splice sites.
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