Male-derived sex-peptide (SP) induces profound changes in the behaviour of Drosophila females, resulting in decreased receptivity to further mating and increased egg laying. SP can mediate the switch in female reproductive behaviours via a G protein-coupled receptor, SPR, in neurons expressing fruitless, doublesex and pickpocket. Whether SPR is the sole receptor and whether SP induces the postmating switch in a single pathway has not, to our knowledge been tested. Here we report that the SP response can be induced in the absence of SPR when SP is ectopically expressed in neurons or when SP, transferred by mating, can access neurons through a leaky blood brain barrier. Membrane-tethered SP can induce oviposition via doublesex, but not fruitless and pickpocket neurons in SPR mutant females. Although pickpocket and doublesex neurons rely on G(o) signalling to reduce receptivity and induce oviposition, G(o) signalling in fruitless neurons is required only to induce oviposition, but not to reduce receptivity. Our results show that SP's action in reducing receptivity and inducing oviposition can be separated in fruitless and doublesex neurons. Hence, the SP-induced postmating switch incorporates shared, but also distinct circuitry of fruitless, doublesex and pickpocket neurons and additional receptors.
Alternative splicing is a key feature of human genes, yet studying its regulation is often complicated by large introns. The Down Syndrome Cell Adhesion Molecule (Dscam) gene from Drosophila is one of the most complex genes generating vast molecular diversity by mutually exclusive alternative splicing. To resolve how alternative splicing in Dscam is regulated, we first developed plasmid-based UAS reporter genes for the Dscam variable exon 4 cluster and show that its alternative splicing is recapitulated by GAL4-mediated expression in neurons. We then developed gap-repair recombineering to very efficiently manipulate these large reporter plasmids in Escherichia coli using restriction enzymes or sgRNA/Cas9 DNA scission to capitalize on the many benefits of plasmids in phiC31 integrase-mediated transgenesis. Using these novel tools, we show that inclusion of Dscam exon 4 variables differs little in development and individual flies, and is robustly determined by sequences harbored in variable exons. We further show that introns drive selection of both proximal and distal variable exons. Since exon 4 cluster introns lack conserved sequences that could mediate robust long-range base-pairing to bring exons into proximity for splicing, our data argue for a central role of introns in mutually exclusive alternative splicing of Dscam exon 4 cluster.
Alternative splicing of pre-mRNA is a major mechanism to increase protein diversity in higher eukaryotes. Dscam, the Drosophila homologue of human DSCAM (Down's syndrome cell adhesion molecule), generates up to 38016 isoforms through mutually exclusive splicing in four variable exon clusters. This enormous molecular diversity is functionally important for wiring of the nervous system and phagocytosis of invading pathogens. Current models explaining this complex splicing regulation include a default repressed state of the variable exon clusters to prevent the splicing together of adjacent exons, the presence of RNA secondary structures important for the release of one specific variable exon from the repressed state and combinatorial interaction of RNA-binding proteins for choosing a specific exon.
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