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. 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.

RNA结合蛋白（RNA-binding protein）性别致死（sex-lethal，Sxl）经典上被定义为黑腹果蝇（Drosophila melanogaster）性别决定与RNA剪接的核心调控因子。然而，该功能并非在所有物种中均保守，且其超出经典通路的额外功能仍未得到广泛探索。本研究在黑腹果蝇大脑中揭示了Sxl在染色质层面的非剪接依赖性功能。通过在幼虫神经元中开展靶向DamID（Targeted DamID，TaDa）谱型分析，我们鉴定发现Sxl可广泛招募至启动子区域，且该过程不依赖于性别与RNA结合活性。值得注意的是，Sxl的染色质占据与RNA聚合酶III（RNA Polymerase III）亚基Polr3E（RPC37）几乎完全重叠，且当Polr3E被敲低时，Sxl的结合会被完全消除。在成熟雄性神经元中敲低Sxl同样会引发广泛的转录变化，尤其富集于代谢基因，同时可改善衰老过程中的负趋地性（negative geotaxis）表型，该表型与Polr3E敲低的表型高度相似。反之，过表达SxlRAC亚型则会增强tRNA合成并上调代谢基因的表达。综上，这些发现揭示了Sxl此前未被认知的功能：通过Polr3E调控RNA聚合酶III活性，进而促进tRNA合成并维持神经元代谢。鉴于RNA聚合酶III调控与神经元衰老之间的新兴关联，本研究凸显Sxl为神经元稳态的新型调控因子。本研究包含多组RNA测序实验，用于检测通过RNAi敲低Sxl与Polr3E时，以及异位/过表达SxlRAC或携带RNA结合位点突变的SxlRNA时的转录变化。上述RNAi诱导与异位/过表达操作均在黑腹果蝇雄性成体神经元中进行，随后收集全脑组织进行RNA测序。