Autoregulation of transformer-2 Alternative Splicing Is Necessary for Normal Male Fertility in Drosophila

Abstract In the male germline of Drosophila the transformer-2 protein is required for differential splicing of pre-mRNAs from the exuperantia and att genes and autoregulates alternative splicing of its own pre-mRNA. Autoregulation of TRA-2 splicing results in production of two mRNAs that differ by the splicing/retention of the M1 intron and encode functionally distinct protein isoforms. Splicing of the intron produces an mRNA encoding TRA-2226, which is necessary and sufficient for both male fertility and regulation of downstream target RNAs. When the intron is retained, an mRNA is produced encoding TRA-2179, a protein with no known function. We have previously shown that repression of M1 splicing is dependent on TRA-2226, suggesting that this protein quantitatively limits its own expression through a negative feedback mechanism at the level of splicing. Here we examine this idea, by testing the effect that variations in the level of tra-2 expression have on the splicing of M1 and on male fertility. Consistent with our hypothesis, we observe that as tra-2 gene dosage is increased, smaller proportions of TRA-2226 mRNA are produced, limiting expression of this isoform. Feedback regulation is critical for male fertility, since it is significantly decreased by a transgene in which repression of M1 splicing cannot occur and TRA-2226 mRNA is constitutively produced. The effect of this transgene becomes more severe as its dosage is increased, indicating that fertility is sensitive to an excess of TRA-2226. Our results suggest that autoregulation of TRA-2226 expression in male germ cells is necessary for normal spermatogenesis.