miR-133 activates notch signalling by repressing Synaptobrevin to control ligand localisation in Drosophila

Precise regulation of Notch signalling is essential for proper eye development in Drosophila. Although the core components of Notch signalling are well characterised, the mechanisms modulating ligand activity remain less understood. Here, we identified microRNA miR-133 as a positive regulator of Notch signalling in a Drosophila eye model. In a Notch-sensitised background, miR-133 overexpression rescued the small-eye phenotype induced by fringe (fng), while miR-133 knockdown exacerbated this defect. miR-133 directly targets Synaptobrevin (Syb), a vesicle-associated SNARE protein, through a conserved site in its 3'UTR. Loss of Syb phenocopied miR-133 overexpression, whereas Syb overexpression enhanced Notch-related defects. miR-133 and Syb exhibited complementary expression patterns, and both miR-133 overexpression and Syb knockdown induced non-autonomous activation of Notch targets and accumulation of Delta and Serrate ligands at the plasma membrane. Furthermore, we show that the E3 ligase Godzilla (Gzl) and Rab11-dependent recycling are essential for Notch activation. Together, our findings revealed a miR-133/Syb/gzl/Rab11 regulatory axis that enhances Notch signalling by modulating ligand trafficking and distribution during development.