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-甲基腺苷（N6-methyladenosine，m6A）是真核生物中分布最广泛的RNA修饰方式，可调控真核生物的RNA命运。保幼激素（Juvenile Hormone，JH）是昆虫不可或缺的内源激素，对昆虫的发育、生长及生理稳态具有关键调控作用。然而，m6A修饰与JH之间的关联机制至今仍未阐明。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）内的m6A相关基因可受JH特异性调控。对家蚕后部丝腺开展m6A测序后发现，大量携带m6A修饰的基因参与mRNA稳定性维持、翻译过程及mRNA降解调控。GO富集分析结果显示，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通路。通过联合分析转录组测序与m6A测序数据，本研究证实JH可显著调控家蚕后部丝腺中mRNA的m6A修饰水平，该调控作用尤其体现在丝相关基因中，包括丝胶蛋白2（sericin2）、丝抑素1（seroin1）、BmSPI5、BmSPI4、Ldb等。此外，本研究发现敲低METTL3的表达后，丝相关基因的转录水平显著改变，验证了丝相关基因是m6A修饰的靶标分子。通过在BmN细胞中对m6A修饰位点进行诱变实验，本研究进一步证实，BmSPI4与BmSPI5的m6A修饰可介导JH对这两个基因表达的增强效应。综上，本研究结果表明，m6A修饰广泛存在于家蚕后部丝腺的mRNA中，并揭示了一种全新的表观遗传调控机制，介导JH对家蚕后部丝腺的生物学功能调控，尤其是在蚕丝生产相关过程中。本研究为解析m6A修饰在昆虫中的功能提供了全新的研究视角。