Juvenile hormone regulates silk gene expression by m⁶A RNA methylation

Juvenile hormone (JH) is an indispensable insect hormone that is critical in regulating insect development and physiology. N6-methyladenosine (m⁶A) is the most abundant modification of RNA that regulates RNA fate in eukaryotic organisms. However, the relationship between m⁶A and JH remains largely unknown. Here, we found that the application of a Juvenile hormone analog (JHA) extended the larval period of Bombyx mori and increased the weight and thickness of the cocoon. Interestingly, global transcriptional patterns revealed that m⁶A-related genes are specifically regulated by JHA in the posterior silk gland (PSG) that synthesizes the major component of cocoon silk. By transcriptome and m⁶A sequencing data conjointly, we discovered that JHA significantly regulated the m⁶A modification in the PSG of B. mori and many m⁶A-containing genes are related to nucleic acid binding, nucleus, and nucleobase-containing compound metabolism. Notably, 547 genes were significantly regulated by JHA at both the m⁶A modification and expression levels, especially 16 silk-associated genes, including sericin2, seroin1, Serine protease inhibitors 4 (BmSPI4), Serine protease inhibitors 5 (BmSPI5), and LIM domain-binding protein 2 (Ldb). Among them, 11 silk associated genes were significantly affected by METTL3 knockdown, validating that these genes are targets of m⁶A modification. Furthermore, we confirm that JHA directly regulates the expression of BmSPI4 and BmSPI5 through m⁶A modification of CDS regions. These results demonstrate the essential role of m⁶A methylation regulated by JH in PSG, and elucidate a novel mechanism by which JH affects silk gland development via m⁶A methylation. This study uncovers that m⁶A modification is a critical factor mediating the effect of JH in insects.