Juvenile hormone regulates silk-associated genes expression in Bombyx mori though m6A modification.

N6-methyladenosine (m6A) is the most abundant modification of RNA that regulates RNA fate in eukaryotic organisms. Juvenile Hormone (JH) is an indispensable insect hormone that is critical in regulating insect development, growth, and physiology. However, the relationship of m6A and JH remains an enigma. Here, global transcriptional patterns revealed that m6A-related genes are specifically regulated by JH in the posterior silk gland (PSG) of Bombyx mori. m6A sequencing of silkworm PSG found that many m6A-containing genes are involved in mRNA stability, translation, and degradation. GO enrichment analysis revealed that m6A-containing genes are relating to nucleic acid binding, nucleus, and nucleobase-containing compound metabolism. By analyzing transcriptome sequencing and m6A sequencing data conjointly, we discovered that JH significantly regulated the m6A modification of mRNA in PSG of B. mori, especially in silk-associated genes including sericin2, seroin1, BmSPI5, BmSPI4, Ldb, etc. Moreover, we found that the expression of silk-associated genes of are significantly regulated by METTL3 knockdown, validating silk-associated genes are targets of m6A modification. We identified that m6a modification of BmSPI4 and BmSPI5 mediates the JH-enhancing expression of BmSPI4 and BmSPI5 though mutagenesis of m6A sites in BmN cells. Taken together, these results suggest that m6a modification widely distributes the mRNA of silkworm PSG and plays a novel epigenetic mechanism for mediating JH effects on PSG of B. mori, particularly in silk production. Our findings provide a novel insight into the role of m6A in insects.

N6-甲基腺嘌呤（N6-methyladenosine，m6A）是真核生物中调控RNA命运的最丰富RNA修饰类型。保幼激素（Juvenile Hormone，JH）是一类不可或缺的昆虫激素，在调控昆虫发育、生长及生理稳态过程中发挥关键作用。然而，m6A与JH之间的关联至今仍是一个未解之谜。本研究通过全局转录组分析发现，家蚕（Bombyx mori）后部丝腺（posterior silk gland，PSG）中的m6A相关基因受JH特异性调控。对家蚕后部丝腺进行m6A测序后发现，大量携带m6A修饰的基因参与mRNA稳定性维持、翻译及降解过程。基因本体富集分析（GO enrichment analysis）显示，携带m6A修饰的基因显著富集于核酸结合、细胞核及含核碱基化合物代谢等功能条目。通过联合分析转录组测序与m6A测序数据，我们证实JH可显著调控家蚕后部丝腺中mRNA的m6A修饰水平，该调控效应在丝相关基因中尤为显著，涉及丝胶蛋白2（sericin2）、丝抑素1（seroin1）、BmSPI5、BmSPI4、Ldb等基因。此外，我们发现敲低甲基转移酶样3（METTL3）可显著改变丝相关基因的表达水平，验证了丝相关基因是m6A修饰的靶标分子。通过在家蚕BmN细胞（BmN cells）中对m6A位点进行诱变，我们证实BmSPI4与BmSPI5的m6A修饰可介导JH对这两个基因表达的增强作用。综上，上述结果表明，m6A修饰广泛分布于家蚕后部丝腺的mRNA转录本中，并揭示了一种介导JH调控家蚕后部丝腺功能的全新表观遗传机制，该机制在蚕丝生产相关生物学过程中发挥重要作用。本研究为理解m6A在昆虫中的生物学功能提供了新的研究视角。