The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster

 In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2)d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA. Received:23 May 1996 Accepted:3 July 1996     

Tribolium castaneum Transformer-2 regulates sex determination and development in both males and females

Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes.     

The gene fl(2)d is required for various Sxl-controlled processes in Drosophila females

Summary In Drosophila melanogaster, the gene Sex-lethal (Sxl) controls the processes of sex determination, dosage compensation, oogenesis and sexual behaviour. The control of Sxl is by alternative splicing of its primary RNA. We have identified a gene, female-lethal-2-d (fl(2)d), which is needed for the female-specific splicing of Sxl RNA and which also has a vital function independent of Sxl. Here we analyse other aspects of the gene fl(2)d. Specifically, we have analysed the effect of the temperature-sensitive mutation fl(2)d ¹ on the viability of adult flies homozygous for this mutation. We have found that the viability of the mutant females is reduced, while that of the mutant males is not affected. In addition, the capacity of the mutant females to be inseminated is considerably reduced, whilst all the mutant males are able to inseminate females. These effects on females are suppressed by Sxl ^M1. However, the fat body cells of fl(2)d ¹ homozygous females are able to synthesize yolk proteins at the restrictive temperature. We have also carried out, in males, a clonal analysis of fl(2)d ², a mutation lethal in both sexes. We have found that the clones are fully viable. We conclude that the gene fl(2)d seems to be necessary during the adult life of females for the processes that require Sxl ⁺ activity. Moreover, the Sxl-independent vital function of fl(2)d seems to be required in both sexes only during larval development. Offprint requests to: L. Sánchez     

Regulation of Sex-Specific Selection of fruitless 5′ Splice Sites by transformer and transformer-2

In Drosophila melanogaster, the fruitless (fru) gene controls essentially all aspects of male courtship behavior. It does this through sex-specific alternative splicing of the fru pre-mRNA, leading to the production of male-specific fru mRNAs capable of expressing male-specific fru proteins. Sex-specific fru splicing involves the choice between alternative 5′ splice sites, one used exclusively in males and the other used only in females. Here we report that the Drosophila sex determination genes transformer (tra) and transformer-2 (tra-2) switch fru splicing from the male-specific pattern to the female-specific pattern through activation of the female-specific fru 5′ splice site. Activation of female-specific fru splicing requires cis-acting tra and tra-2 repeat elements that are part of an exonic splicing enhancer located immediately upstream of the female-specific fru 5′ splice site and are recognized by the TRA and TRA-2 proteins in vitro. This fru splicing enhancer is sufficient to promote the activation by tra and tra-2 of both a 5′ splice site and the female-specific doublesex (dsx) 3′ splice site, suggesting that the mechanisms of 5′ splice site activation and 3′ splice site activation may be similar.     

The translation initiation factor eIF4E regulates the sex-specific expression of the master switch gene Sxl in Drosophila melanogaster

In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors--the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d--that have been directly implicated in Sxl splicing regulation.     

Requirement offlex (femalelethal onX) in the development of the female germ line ofDrosophila melanogaster

Drosophila melanogaster females homozygous forflex, an X-linked recessive mutation, do not survive. Hemizygous males are unaffected. Homozygous embryos appear to lack SXL, the product of theSex-lethal (Sxl) gene, apparently as a result of disruption ofSxl splicing. It is known that bothSxl and its somatic splicing regulators [snf andfl(2)d] also function in the development of the female germ line. For this reason, we investigated the role offlex in the germ line by generatingflex/flex clones inflex/+ females. Females carrying such clones in their germ lines do not lay eggs whereas females carryingflex+ eggs lay viable eggs. Additionally, DAPI staining of ovarioles showed that diploid germ cells that are homozygous mutant forflex do not complete oogenesis. These results indicate that theflex+ gene product may be required for the development of the female germ line.     

Sxl in the germline of Drosophila: A target for somatic late induction

In Drosophila, the sex of germ cells is determined by autonomous and inductive signals. Somatic inductive signals can drive XX germ cells into oogenesis or into spermatogenesis. An autonomous signal makes XY germ cells male and unresponsive to sex determination by induction. The elements forming the X:A ratio in the soma and the genes tra, tra2, dsx, and ix that determine the sex of somatic cells have no similar role in the germline. The gene Sxl, however, is required for female differentiation of somatic and germ cells. Inductive signals that are dependent on somatic tra and dsx expression already affect the sex-specific development of germ cells of first instar larvae. At this early stage, however, germline expression of Sxl does not appear to affect the sexual characteristics of germ cells. Since inductive signals dependent on tra and dsx nevertheless influence the choice of sex-specific splicing of Sxl, it can be concluded that Sxl is a target of the inductive signal, but that its product is required late for oogenesis. Other genes must therefore control the early sexual dimorphism of larval germ cells. © 1994 Wiley-Liss, Inc.     

Structure and Novel Functional Mechanism of Drosophila SNF in Sex-Lethal Splicing

Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B″, and it is essential in Drosophila sex determination. We found that, besides its ability to bind U1 snRNA, SNF can also bind polyuridine RNA tracts flanking the male-specific exon of the master switch gene Sex-lethal (Sxl) pre-mRNA specifically, similar to Sex-lethal protein (SXL). The polyuridine RNA binding enables SNF directly inhibit Sxl exon 3 splicing, as the dominant negative mutant SNF¹⁶²¹ binds U1 snRNA but not polyuridine RNA. Unlike U1A, both RNA recognition motifs (RRMs) of SNF can recognize polyuridine RNA tracts independently, even though SNF and U1A share very high sequence identity and overall structure similarity. As SNF RRM1 tends to self-associate on the opposite side of the RNA binding surface, it is possible for SNF to bridge the formation of super-complexes between two introns flanking Sxl exon 3 or between a intron and U1 snRNP, which serves the molecular basis for SNF to directly regulate Sxl splicing. Taken together, a new functional model for SNF in Drosophila sex determination is proposed. The key of the new model is that SXL and SNF function similarly in promoting Sxl male-specific exon skipping with SNF being an auxiliary or backup to SXL, and it is the combined dose of SXL and SNF governs Drosophila sex determination.     

Establishment of a novel in vivo sex-specific splicing assay system to identify a trans-acting factor that negatively regulates splicing of Bombyx mori dsx female exons

The Bombyx mori homolog of doublesex, Bmdsx, plays an essential role in silkworm sexual development. Exons 3 and 4 of Bmdsx pre-mRNA are specifically excluded in males. To explore how this occurs, we developed a novel in vivo sex-specific splicing assay system using sexually differentiated cultured cells. A series of mutation analyses using a Bmdsx minigene with this in vivo splicing assay system identified three distinct sequences (CE1, CE2, and CE3) positioned in exon 4 as exonic splicing silencers responsible for male-specific splicing. Gel shift analysis showed that CE1 binds to a nuclear protein from male cells but not that from female cells. Mutation of UAA repeats within CE1 inhibited the binding of the nuclear protein to the RNA and caused female-specific splicing in male cells. We have identified BmPSI, a Bombyx homolog of P-element somatic inhibitor (PSI), as the nuclear factor that specifically binds CE1. Down-regulation of endogenous BmPSI by RNA interference significantly increased female-specific splicing in male cells. This is the first report of a PSI homolog implicated in the regulated sex-specific splicing of dsx pre-mRNA.