A miR-305-5p/Nerfin-1/Cyp18a1 cascade fine-tunes 20-Hydroxyecdysone degradation for precise pupariation in Drosophila

Precise steroid hormone pulse dynamics, governed by biosynthesis and degradation, serve as critical temporal signals coordinating animal life-stage transitions. While pulse initiation is well-studied, mechanisms enabling rapid termination and clearance remain unclear. Here, focusing on insect metamorphosis, orchestrated by 20-hydroxyecdysone (20E) pulses, we identify transcription factor Nervous fingers 1 (Nerfin-1) as a central coordinator of the steroid hormone homeostasis in Drosophila melanogaster. We demonstrate dynamic nerfin-1 expression in the prothoracic gland (PG) and fat body, tightly correlating with 20E titers. PG-specific knockdown of nerfin-1 causes developmental arrest and reduced ecdysteroid levels; these defects are rescued by dietary 20E supplementation. Transcriptomic and chromatin immunoprecipitation analyses reveal that Nerfin-1 directly represses cyp18a1, a key enzyme for 20E degradation, thereby delaying hormone clearance during the prepupal stage. Furthermore, we identify miR-305-5p as a critical upstream post-transcriptional regulator of nerfin-1. miR-305-5p exhibits an inverse temporal expression pattern relative to nerfin-1 and directly targets its 3′UTR to suppress translation. Overexpression of miR-305-5p in the PG reduces nerfin-1 levels, elevates cyp18a1 expression, depletes ecdysteroid titers, and delays development. Crucially, these phenotypes are rescued by either dietary 20E or cyp18a1 knockdown, confirming pathway integrity. Collectively, our findings define a novel miR-305-5p/Nerfin-1/Cyp18a1 regulatory cascade that fine-tunes hormone pulse dynamics to ensure robust developmental progression.