Diapause termination alters the m⁶A methylation profiles and regulates gene expression associated with early...

Univoltine and bivoltine silkworms (Bombyx mori) exhibit typical diapause characteristics. N⁶-methyladenosine (m⁶A), an important epigenetic modification of RNAs in eukaryotic cells, plays a vital role in developmental regulation. In this study, we aimed to explore whether m⁶A modification regulates gene expression associated with early embryonic development in the silkworm. Hydrochloric acid (HCl) treatment was used to terminate silkworm embryonic diapause. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were used to identify genes with m⁶A modifications associated with early embryonic development. By combining MeRIP-seq and RNA-seq data, we identified 56 differentially expressed genes (DEGs) with distinct changes in messenger RNA (mRNA) m⁶A modification, predominantly in the Hippo pathway and amino sugar and nucleotide sugar metabolism. In the Hippo pathway, the cadherin-related tumor suppressor (Ft), extracellular serine/threonine protein kinase four-jointed (Fj), Kibra, and Dachsous (Ds) genes showed less m⁶A modification of their mRNA after HCl treatment and downregulated expression. Additionally, genes downstream of the Hippo pathway, the FERM domain containing expanded, serine/threonine-protein kinase hippo, scaffold protein Salvador, and serine/threonine-protein kinase warts were also downregulated. We speculate that the downregulation of the key genes Ft, Fj, Ds, and Kibra by m⁶A modification leads to decreased expression of downstream genes, which may affect the transcriptional coactivator Yorkie and inhibit the Hippo pathway after diapause termination, thereby promoting early silkworm embryonic development. Overall, our findings provide novel insight into the systematic function of RNA methylation in regulating embryogenesis and the importance of m⁶A modification in the developmental process in the silkworm.