GATOR1复合体调控果蝇卵细胞减数分裂进程的分子机制研究

Target of Rapamycin Complex 1 (TORC1) is a master regulator that mediates nutrient status and cell growth. Previously, we found that the GATOR1 complex, comprised of the Nprl2, Nprl3 and Iml1 proteins, is an inhibitor of TORC1 and plays important roles in meiotic entry and cell survival under starvation. However, the molecular mechanism of GATOR1 on meiotic cell cycle is little known. Meiosis promotes genomic diversity through homologous recombination, a process require DNA double-stranded breaks (DSBs). On the other hand, DSBs represent a dangerous form of DNA damage that can result in dramatic and permanent changes to the genome. Thus, the DSB formation must be exquisitely controlled during gamete formation. Here our preliminary data show that GATOR1 mutant oocytes accumulate an increased number of DSBs and activity of p53 in early prophase of meiosis I. These results suggest that the GATOR1 is required for maintaining genomic stability. This project will study the DSB, p53 activity, homologous chromosomes recombination rate and transposable element (TE) expression in Drosophila oocyte to elucidate the molecular function of GATOR1 complex. Through inhibiting meiotic DSB machines, TORC1 activity and p53 pathway, the mechanism of GATOR1 regulating DSB formation, p53 activity and cell death during early meiosis will be determined. This project will elucidate the link between the metabolic pathway and genomic stability in germ line. This work will provide new idea for insect reproduction control and theoretical support for mammal meiosis mechanism.

TORC1是调节细胞生长和代谢的关键通路。GATOR复合体通过动态控制TORC1活性，调节果蝇卵细胞的生长与分化，但对其如何在分子水平调节减数分裂进程仍不清楚。DNA双链断裂（DSB）是同源染色体重组的基础，但过多的DSB会引起基因组的不稳定。本项目以GATOR1复合体的组分蛋白Nprl2、Nprl3、Iml1的基因突变和转基因果蝇为对象，通过研究GATOR1突变体卵细胞中的DSB水平、p53活性、同源重组效率、转座子活化等，阐明GATOR1复合体的分子功能；通过研究减数分裂DSB的生成与修复、TORC1活性、p53通路等对GATOR1突变体卵细胞中DSB水平、p53活性及细胞凋亡的影响，阐明GATOR1复合体保护早期卵细胞的机制。本项目将揭示GATOR1复合体在昆虫卵细胞基因组稳定中的作用，为干预昆虫生殖提供新思路，也为研究高等动物减数分裂机制提供理论支持。

在昆虫卵母细胞发育的减数分裂阶段，来自父本和母本染色体之间的同源重组增加卵子遗传物质的多样性，为后代进化和适应环境提供基础。DNA双链断裂（DSB）产生和修复是实现同源染色体交换的基础，但过多的DSB会引起基因组的不稳定，导致细胞凋亡。因此，DSB发生的时间和水平被严格控制。我们前期发现GATOR1复合体组分蛋白Nprl2、Nprl3或Iml1的缺失会引起果蝇卵细胞中DSB水平增加。本项目对GATOR1复合体调控卵细胞中DSB水平的作用机制及生物学功能进行了深入研究。我们发现GATOR1复合体通过抑制控制细胞生长和代谢的关键通路TORC1的活性，促进减数分裂进程中DSB的修复，使卵细胞中DSB数目增加、维持时间延长，但并不影响减数分裂DSB的产生。GATOR1突变体果蝇卵细胞中存在调控DNA损伤修复和细胞凋亡的关键蛋白p53活性的增加，这是由于GATOR1突变体中的减数分裂DSB水平增加引起的。进一步发现GATOR1和p53具有协同效应，通过作用于不同的信号通路，共同抑制转座子的活化，维持卵细胞基因组的稳定。我们利用免疫共沉淀的方法筛选到FKBP39蛋白可以与GATOR1的结合并促进GATOR1蛋白的泛素化降解，进而影响卵细胞的代谢和发育。本项目揭示了GATOR1 复合体和TORC1通路在昆虫卵细胞基因组稳定中的作用，探索了GATOR1蛋白的稳定性调节机制，为通过细胞代谢机制干预昆虫生殖提供新思路，也为研究高等动物中的减数分裂机制提供理论支持。