Sex-lethal is recruited to chromatin to promote neuronal tRNA synthesis in males through RNA Polymerase III regulation [RNA-seq]

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. Overall design: These are RNAseq experiments to examine transcriptional changes when Sxl and Polr3E are knocked down with RNAi, as well as when SxlRAC or SxlRNA (mutated RNA binding sites) are mis/overexpressed. RNAi and mis/overexpression was induced in Drosophila male adult neurons and then whole heads collected for RNAseq.