The role of the fruitless P3 transcript in development in Drosophila melanogaster

The generation of multiple transcripts from a single gene through alternative promoter usage and alternative splicing is a major source of functional diversity in eukaryotic genomes. The fruitless (fru) gene of Drosophila melanogaster is a classic example of transcript complexity, producing at least 15 transcript isoforms through the use of alternative promoters and terminal exons. While transcripts derived from the sex-specifically spliced P1 promoter have been extensively studied for their roles in sex determination and courtship behaviour, the functions of most non-sex-specific fru transcripts remain poorly understood. Previous studies suggested that transcripts originating from the P3 and/or P4 promoters may contribute to developmental viability, but their individual roles could not be resolved because available mutations disrupted multiple transcript classes simultaneously. Here, we used transcript-specific RNA interference (RNAi) and the Gal4/UAS system to investigate the function of fru P3 transcripts. Loss of P3 expression significantly increased developmental time to pupariation and resulted in complete lethality during pupal development despite normal survival to pupariation. Most mortality occurred during early and mid-pupal stages, indicating a critical requirement for P3-derived transcripts during metamorphosis. Notably, P3 knockdown did not phenocopy complete loss of fruitless, as knockdown individuals survived substantially later into development than fru null mutants, suggesting that additional fruitless transcript classes contribute essential functions during earlier developmental stages. Together, these findings establish an independent developmental role for the fru P3 transcript class and demonstrate that transcript-specific functions contribute substantially to the biological complexity of the fruitless locus.