A miR-305-5p/Nerfin-1/Cyp18a1 cascade fine-tunes 20-hydroxyecdysone degradation to time pupariation in Drosophila

Steroid hormone pulses provide critical temporal signals that synchronize developmental transitions, yet the mechanisms that restrain hormone clearance and preserve pulse timing remain unclear. Using Drosophila melanogaster metamorphosis, a process orchestrated by 20-hydroxyecdysone (20E) pulses, this study identified Nervous fingers 1 (Nerfin-1) as a key transcriptional regulator of 20E dynamics during development. Notably, nerfin-1 displayed stage-dependent expression in the prothoracic gland (PG) and fat body, with temporal patterns closely aligned with 20E titers. PG-specific nerfin-1 knockdown arrested development and reduced ecdysteroid levels, whereas dietary 20E restored developmental progression. Integrated transcriptomic and chromatin immunoprecipitation analyses revealed that Nerfin-1 directly repressed cyp18a1, which encodes a major 20E-inactivating enzyme, thereby delaying hormone clearance during the prepupal stage. Upstream of this regulatory pathway, miR-305-5p acted as a post-transcriptional regulator of nerfin-1, showing an inverse temporal expression pattern and directly targeting the nerfin-1 3′ untranslated region (UTR) to suppress translation. PG-specific miR-305-5p overexpression reduced nerfin-1 levels, increased cyp18a1 expression, depleted ecdysteroid titers, and delayed development. Crucially, these phenotypes were rescued by either dietary 20E supplementation or cyp18a1 knockdown, confirming that altered ecdysteroid degradation underlies these effects. Collectively, these findings define a novel miR-305-5p/Nerfin-1/Cyp18a1 cascade that fine-tunes hormone pulse dynamics to ensure robust developmental progression.