BmADARa-mediated RNA editing regulates silk gland development and silk protein expression via miR-3315 targeting BmSuc1 in Bombyx mori.

The ADAR family, which catalyzes adenosine-to-inosine (A-to-I) RNA editing on double-stranded RNA, represents an evolutionarily conserved RNA-modifying enzyme. While ADAR regulates microRNA (miRNA) maturation through both editing-dependent and -independent mechanisms, its role in organ development remains poorly characterized. In Bombyx mori, we previously identified high expression of BmADARa and BmSuc1 (encoding β-fructofuranosidase) in silk glands, with BmSuc1 known to regulate silk gland development. Here, we demonstrate that BmADARa controls silk gland patterning through miR-3315-BmSuc1 signaling axis. Specifically, we constructed RNAi-BmADARa mutants, and revealed that BmADARa is involved in regulating silk gland development and the expression levels of BmSuc1. Subsequent in-depth investigations demonstrated that BmADARa controls BmSuc1 expression by acting on its 3'UTR. Leveraging miRNA target prediction tools (miRanda and RNAhybrid), we identified miR-3315 as the exclusive candidate targeting the BmSuc1-3'UTR, with additional binding sites detected in the BmSuc1-CDS. BmADARa-RIP assays and Sanger sequencing provided conclusive evidence that BmADARa promotes miR-3315 maturation by editing pri-miR-3315. Moreover, KO-BmSuc1 mutants displayed altered expression patterns of sericin and fibroin genes, further validating that BmADARa regulates silk gland development through BmSUC1. In conclusion, our results show that BmADARa regulates the expression of BmSUC1, thereby positively influencing sericin gene expression in the anterior and middle silk glands and negatively regulating fibroin gene expression in the posterior silk gland. These results offer novel perspectives on the regulatory mechanisms governing silk gland development.