Sex-specific development in haplodiploid honeybee is controlled by the female-embryo-specific activation of thousands of intronic lncRNAs
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ABSTRACT: Sex determination in the honeybee (Apis mellifera) is governed by the queen-controlled unfertilization or fertilization of embryo, though the mechanisms of determination are poorly understood. Here, we obtained the transcriptomes from individual worker and drone embryo during the embryonic development (day 1 to day 3). We show that transcriptional difference between worker and drone embryo is very small during the first day of hatching, during which sex-determinant gene csd expresses similarly. Differential transcription between worker and drone embryo bursts at day 2, among which csd is induced in worker embryo at day 2 and sex-lethal gene sxl is repressed in male embryo. An unexpected global regulation of alternative splicing accompanies the honeybee embryonic development, and male and worker embryo show distinct regulatory patterns and mechanisms. This study suggests the honeybee sex determination is more globally controlled at both the transcriptional and alternative splicing levels.
Project description:Conversely to canonical splicing, back-splicing covalently ligates the upstream 3' splice site (SS) with downstream 5'SS and generates exonic circular RNAs (circRNAs) that are widely-identified in eukaryotes and have regulatory functions in gene expression. However, sex-specific back-splicing in Drosophila has not been investigated and its regulation remains unclear. Here, we performed multiple RNA-seq of various sex-specific Drosophila samples including head, body and gonads from both genders, and identified more than ten thousand of circular RNAs, in which hundreds are sex-differentially expressed and back-spliced. Intriguingly, we found that expression of SXL, an RNA-binding protein encoded by Sex-lethal (Sxl), the master Drosophila sex-determination gene which only functionally spliced in females, promotes back-splicing of many female-differentially expressed circRNAs in the male S2 cells, while expression of a SXL mutant did not. Using a monoclonal antibody, we further obtained the transcriptome-wide RNA-binding sites of SXL through a PAR-CLIP approach and revealed that SXL-binding on flanking exons and introns of pre-mRNAs facilitates back-splicing of those circRNAs, whereas SXL-binding on the circRNA exons inhibits the back-splicing. This study provides strong evidence that SXL has a regulatory role in back-splicing to generate sex-specifc circRNAs, as well as in the initiation of Drosophila sex-determination cascade through canoncial forward-splicing.
Project description:We have identified a honeybee (Apis mellifera) odorant receptor (Or) for the queen substance 9-oxo-2-decenoic acid (9-ODA) from four candidate sex pheromone odorant receptors from the honeybee genome based on their biased expression in drone antennae. Keywords: Tissue Comparison
Project description:Olfaction system plays a fundamental role in mediating insect behavior. Besides, the division of queen, worker and drone, honeybee also exhibit an age-dependent division of labor. Worker bees perform discrete sets of behaviors throughout their lifespan. These behavioral states rely on the sense of the environments and chemical communications via their olfactory system - antennae. However, the olfactory adaption mechanism of workers in these processes of behavioral development is still unclear. In this study, we conducted a comprehensive and quantitative analysis of gene expression in Apis mellifera antenna of newly emerged workers, nurses, foragers, and defenders using RNA-seq. We found that antennae tissues continue to develop after transformation from pupae to adult. Additionally, we identified both developmental and labor-division specific expressed genes. We validated the unexpected discovery of major royal jelly protein genes, which are highly and specifically expressed in nurse honeybee workers. We further identified and validated that significant alternative splicing events are also involved in the development and division of labor. These findings provided a comprehensive transcriptome profile and new perspective into the molecular mechanism underlying honeybee division of labor.
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 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.
Project description:The RNA-binding protein sex-lethal (Sxl) is classically defined as a master regulator of sex determination and RNA splicing in Drosophila melanogaster. However, this role is not conserved in all species and functions beyond this canonical pathway remain largely unexplored. Here, we uncover a splicing-independent function for Sxl at the chromatin level in the Drosophila brain. Using Targeted DamID (TaDa) profiling in larval neurons, we identify widespread recruitment of Sxl to promoter regions, independent of sex and RNA binding activity. Notably, Sxl chromatin occupancy exhibits near-complete overlap with Polr3E (RPC37), an RNA Polymerase III subunit, with Sxl binding abolished upon Polr3E knockdown. Depletion of Sxl in mature male neurons equally induces widespread transcriptional changes, particularly in metabolic genes, and improves negative geotaxis during ageing, phenotypes that closely mirror Polr3E knockdown. Conversely, overexpression of SxlRAC isoform enhances tRNA synthesis and upregulates metabolic gene expression. Together, these findings reveal a previously unrecognised role for Sxl in regulating Pol III activity via Polr3E, promoting tRNA synthesis and supporting neuronal metabolism. Given the emerging tie between Pol III regulation and neuronal ageing, our study highlights Sxl as a novel modulator of neuronal homeostasis.
Project description:The RNA-binding protein sex-lethal (Sxl) is classically defined as a master regulator of sex determination and RNA splicing in Drosophila melanogaster. However, this role is not conserved in all species and functions beyond this canonical pathway remain largely unexplored. Here, we uncover a splicing-independent function for Sxl at the chromatin level in the Drosophila brain. Using Targeted DamID (TaDa) profiling in larval neurons, we identify widespread recruitment of Sxl to promoter regions, independent of sex and RNA binding activity. Notably, Sxl chromatin occupancy exhibits near-complete overlap with Polr3E (RPC37), an RNA Polymerase III subunit, with Sxl binding abolished upon Polr3E knockdown. Depletion of Sxl in mature male neurons equally induces widespread transcriptional changes, particularly in metabolic genes, and improves negative geotaxis during ageing, phenotypes that closely mirror Polr3E knockdown. Conversely, overexpression of SxlRAC isoform enhances tRNA synthesis and upregulates metabolic gene expression. Together, these findings reveal a previously unrecognised role for Sxl in regulating Pol III activity via Polr3E, promoting tRNA synthesis and supporting neuronal metabolism. Given the emerging tie between Pol III regulation and neuronal ageing, our study highlights Sxl as a novel modulator of neuronal homeostasis.
Project description:Transcriptional profiling of brain tissue of was performed by RNA-SEQ in workers, queens and males. The sex and caste specific expressed and spliced genes were examined comparing drone, worker and queen honeybees.
Project description:Protein phosphorylation is known to regulate a wide scenario of key cellular processes. However, phosphorylation mediated regulatory networks in honeybee embryogenesis are largely unknown. We identified 6342 phosphosites from 2438 phosphoproteins and predicted 168 kinases in the honeybee embryo. The worker and drone develop similar phosphoproteome architectures and central phosphorylation events during embryogenesis.