Neuronal protein Sex-lethal modulates tRNA synthesis via the polymerase III subunit Polr3E in male Drosophila neurons

The RNA-binding protein Sex-lethal (Sxl) is classically known as a master regulator of sex determination and mRNA splicing in Drosophila melanogaster. However, this role is not conserved across species, and functions beyond the canonical pathway remain poorly understood. In this study, we uncover a splicing-independent role for Sxl at the chromatin level in the Drosophila brain. Using Targeted DamID (TaDa) profiling in neurons, we identify widespread binding of Sxl to promoter regions, independent of sex or 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 induces widespread transcriptional changes, particularly in metabolic genes, and improves negative geotaxis during aging, phenotypes that closely mirror Polr3E knockdown. Conversely, overexpression of the brain-specific SxlRAC transcript leads to severe climbing deficits and upregulated gene expression associated with metabolism and translation. Manipulating Sxl levels in the brain significantly impacts select tRNA production and global protein synthesis rates. Together, these findings reveal a previously unrecognized role for Sxl in regulating Pol III activity via Polr3E, modulating tRNA synthesis and supporting neuronal metabolism. Given the emerging tie between Pol III regulation and neuronal aging, our study highlights Sxl as a novel factor in neuronal homeostasis.The RNA-binding protein Sex-lethal regulates sex determination and mRNA splicing in Drosophila. This study reveals a new role in neuronal homeostasis and transfer RNA synthesis via Polr3E regulation in neurons.