Identification of an atypical microRNA with sequence variation in the silkworm Bombyx mori

MicroRNAs (miRNAs) play essential roles in posttranscriptional regulation of gene expression. During miRNA biosynthesis, miRNA precursors are cleaved by Dicer at their loop structure to generate mature miRNAs. However, the functional consequences of sequence alterations in the loop region remain poorly understood. The silkworm Bombyx mori is a lepidopteran insect with many genetic strains. In a B. mori strain exhibiting translucent larval skin, we unexpectedly identified a mutant form of miR-3260 in which part of the predicted loop structure was deleted. Here, we aimed to analyze the function of wild-type miR-3260 and the impact of this loop structure mutation in B. mori. First, we predicted potential binding partners for wild-type miR-3260 using the RNAhybrid tool and identified eight candidates, including two juvenile hormone (JH)-related genes. Expression analysis revealed high miR-3260 expression in the corpora allata. Furthermore, treatment of BmN cells with JH led to upregulated miR-3260 expression, suggesting a relationship between miR-3260 and JH signaling. Although miR-3260 mimics and inhibitors altered the expression of two JH-related candidate genes, the observed changes were not consistent with the predicted regulatory effects of miR-3260 , and no observable larval phenotype was detected. Additionally, analysis of Dicer processing showed that neither the wild-type nor mutant miR-3260 precursor underwent Dicer-mediated cleavage. Although miR-3260 responds to JH, its function as a conventional miRNA was not supported. These findings suggest that miR-3260 may act as a JH-responsive non-coding RNA or represent a novel atypical RNA distinct from canonical miRNAs.