Caprin2通过结合Mettl14调控线粒体Drp1的m6A修饰在卵母细胞体外成熟中的作用和机制研究

翻译起始在卵母细胞恢复减数分裂过程中受到精密的调控，是卵母细胞体外成熟（IVM）中的关键事件，然而IVM的翻译调控机制并未阐明。通过卵母细胞蛋白组数据分析，我们发现Caprin2在卵母细胞成熟过程中存在显著的下调表达。在人卵母细胞中干预Caprin2促进了卵母细胞体外成熟。Caprin2下调抑制了线粒体Drp1的表达，进而调控了线粒体自噬和ATP合成。机制研究发现，Caprin2调控了Drp1的m6A甲基化修饰，影响了Drp1的翻译。鉴于Caprin2同工酶Caprin1可以招募Mettl3/Mettl14调控RNA甲基化，并且Drp1存在Mettl14结合位点。因此我们提出假设：Caprin2是否通过结合Mettl14调控了Drp1的RNA甲基化，进而调控了Drp1的翻译，促进了卵母细胞的体外成熟？本项目拟采用免疫共沉淀、MERIP和基因敲除等方法，为卵母细胞翻译起始调控提供理论依据。

Post-transcriptional translation initiation are precisely regulated in the process of oocyte meiosis recovery, to play key roles in oocyte maturation in vitro(IVM). However, the translation regulation mechanism in IVM has not been clarified. Through the analysis of proteomics data of oocytes, we found that Caprin2 was significantly down-regulated during oocyte maturation. Caprin2 protein degradation in human oocytes promoted oocyte maturation in vitro. After Caprin2 intervention, we found that the expression level of Drp1, a key protein for mitochondrial fission, was significantly down-regulated, which further inhibited mitochondrial autophagy and promoted ATP production. Further mechanism studies showed that Caprin2 affected the protein expression of Drp1 by regulating the translation process, and Caprin2 regulated the RNA methylation level of Drp1. Studies have shown that Carpin1, as Caprin2 isoenzyme, achieved specific regulation of RNA methylation by binding Mettl3/Mettl14 and target RNA. Based on database, we found that Drp1-RNA may bind to Mettl14. Therefore, we proposed a hypothesis: whether Caprin2 specifically regulates the RNA methylation of mitochondrial Drp1 by binding Mettl14, thereby regulating the translation of Drp1 and promoting the maturation of oocytes in vitro? Methods of immunoprecipitation, MERIP and gene knockout would be used in this project to provide support for the regulation of oocyte translation initiation.