Project description:Sex-lethal (Sxl), a feminizing switch gene in Drosophila, also generates male-specific proteins and non-sex-specific mRNAs, despite the fact that deleting the gene from males causes no obvious phenotypic abnormalities. We specifically investigate how Sxl may affect gene expression in the male head, where SXL isoforms have been detected. The information may help us understand how Sxl acquired its functional roles in sex determination during evolution in Drosophila Keywords: Comparative expression, genetic modification
Project description:In Drosophila female development is governed by a single RNA-binding protein, Sex lethal (Sxl), that controls the expression of key factors involved in dosage compensation, germline homeostasis and the establishment of female morphology and behaviour. Functional Sxl protein is only synthesized in female flies. Its expression is established in the pre-cellular, female embryo by an X-chromosome counting mechanism and maintained by a positive feedback loop with Sxl controlling alternative splicing of its own transcript. In male flies this feedback loop is not activated, keeping Sxl expression in the ‘off’ state. But what protects males against accidental triggering of the self-sustaining feedback loop and Sxl protein production snowballing out of control? Here we identify Sister of sex lethal (Ssx) as a protein that antagonizes Sxl in autoregulatory splicing by competition for the same RNA elements. Male flies mutant for ssx exhibit a low level of productive Sxl mRNA splicing and in cultured Drosophila cells, Sxl-induced changes in alternative splicing can be reverted by the expression of Ssx. In sum, this demonstrates that Ssx helps to establish a stable, male-specific gene expression pattern by protecting male flies against triggering Sxl expression.
Project description:In Drosophila female development is governed by a single RNA-binding protein, Sex lethal (Sxl), that controls the expression of key factors involved in dosage compensation, germline homeostasis and the establishment of female morphology and behaviour. Functional Sxl protein is only synthesized in female flies. Its expression is established in the pre-cellular, female embryo by an X-chromosome counting mechanism and maintained by a positive feedback loop with Sxl controlling alternative splicing of its own transcript. In male flies this feedback loop is not activated, keeping Sxl expression in the ‘off’ state. But what protects males against accidental triggering of the self-sustaining feedback loop and Sxl protein production snowballing out of control? Here we identify Sister of sex lethal (Ssx) as a protein that antagonizes Sxl in autoregulatory splicing by competition for the same RNA elements. Male flies mutant for ssx exhibit a low level of productive Sxl mRNA splicing and in cultured Drosophila cells, Sxl-induced changes in alternative splicing can be reverted by the expression of Ssx. In sum, this demonstrates that Ssx helps to establish a stable, male-specific gene expression pattern by protecting male flies against triggering Sxl expression.
Project description:The Drosophila sex determination hierarchy consists of a splicing cascade with sex-specific transcription directing somatic sexual dimorphism. Our understanding of this pathway, and many others, is incomplete. Here we pioneer an approach to expand our knowledge of gene regulatory networks (GRNs) by leveraging natural genetic variation. This approach is generalizable to any natural population, including humans. Two studies from Drosophila female head tissue were used – the DSPR collection (alleles from 15 natural variants) and F1-hybrid collection (alleles from heterozygotes of 75 isogenic lines crossed to w1118) – in a structural equation model (SEM) analysis. We expanded the sex hierarchy GRN by adding novel links among genes in the pathway and by adding novel genes to the pathway. A link from fruitless (fru) to Sex-lethal (Sxl) was found in both populations, which is supported by the presence of fru binding sites in the Sxl locus. The splicing factors male-specific lethal 2 and Rm62 were correctly identified as downstream targets of Sxl. There were 754 additional candidate genes for an expanded sex hierarchy GRN. These candidates were enriched for genes with sex-biased splicing and many components of the spliceosome were placed in the GRN. As with other population-genetic analyses, the number of alleles limits the number of observable interactions. Network expansion was only clear in the F1-hybrid population, which has an average of twice as many alleles as the DSPR population. Independent studies of doublesex and transformer mutants support many novel connections, including evidence for a link between the sex hierarchy and metabolism, with the inclusion of Insulin-like receptor in the sex hierarchy GRN. RNA sequencing was performed on mRNA derived from adult male or female heads, for a total of 9 samples. These samples included females that produce the male isoform of dsx [w/+;DsxD/dsxm+r15 (XX)], and two dsx mutants: females [w/+; dsxm+r15/dsxd+r3 (XX)] and males [w;dsxm+r15/dsxd+r3 (XY)]. Two wild type genotypes (Berlin and Canton-S) were sequenced at the same time, but have previously been published as part of GSE50515. There were at least 3 replicates from biological samples.
Project description:The Drosophila polyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls axon projection, locomotion, and memory. Here we define an unexpectedly specific role for Nab2 in regulating splicing of ~150 exons/introns in the head transcriptome and link the most prominent of these, female retention of a male-specific exon in the sex determination factor Sex-lethal (Sxl), to a role in m6A-dependent mRNA splicing. Genetic evidence indicates that aberrant Sxl splicing underlies multiple phenotypes in Nab2 mutant females. At a molecular level, Nab2 associates with Sxl pre-mRNA and ensures proper female-specific splicing by preventing m6A hypermethylation by Mettl3 methyltransferase. Consistent with these results, reducing Mettl3 expression rescues developmental, behavioral and neuroanatomical phenotypes in Nab2 mutants. Overall these data identify Nab2 as a required regulator of m6A-regulated Sxl splicing and imply a broader link between Nab2 and Mettl3-regulated brain RNAs.