Autoregulation of transformer-2 alternative splicing is necessary for normal male fertility in Drosophila.

M Elaine Mcguffin,Darshna Somaiya,Brigitte Dauwalder,William Mattox,Dawn Chandler

doi:10.1093/genetics/149.3.1477

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-2(226), 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-2(179), a protein with no known function. We have previously shown that repression of M1 splicing is dependent on TRA-2(226), 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-2(226) 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-2(226) 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-2(226). Our results suggest that autoregulation of TRA-2(226) expression in male germ cells is necessary for normal spermatogenesis.

Highlights

THE RNA-binding protein transformer-2 affects alternative splicing of RNAs from several genes with critical roles in Drosophila sexual differentiation
The ratio of alternative male-specific tra-2 mRNAs is affected by gene dosage: If autoregulation of splicing serves to place an upper limit on the amount of TRA2226 encoding mRNA produced, we expected that the ratio of TRA-2179 to TRA-2226 RNAs should increase as the number of transcribed copies of the tra-2ϩ gene is increased
We conclude that male germ cells respond to an increase in the level of tra-2 premRNA by increasing repression of M1 splicing

Summary

Introduction

THE RNA-binding protein transformer-2 (tra-2) affects alternative splicing of RNAs from several genes with critical roles in Drosophila sexual differentiation. In the absence of functional TRA-2, male germ cells appear to initiate spermatogenesis normally but form spermatids with unelongated nuclei that are not motile (Belote and Baker 1983) As a result, such mutant males are sterile. Analysis of transgenic strains that express either TRA2226 or TRA-2179 individually indicated that, while TRA2226 is necessary and sufficient for both sex-specific exu splicing and male fertility, TRA-2179 has no effect on either function (Mattox et al 1996) This indicates that TRA-2226 is the primary functional product and that autoregulation of M1 splicing is part of a negative feedback pathway that serves to quantitatively limit the amount of RNA encoding functional protein (TRA-2226). We find that feedback regulation of this mRNA in the male germline is required for nomal male fertility

Methods

Results

Conclusion