RPB1羧基端磷酸化在小鼠卵母细胞染色体分离中的作用和分子机制

In oocyte meiosis, chromosome segregation errors could lead to embryo aneuploid, which is the main cause of infertility, spontaneous abortion and birth defects. Accuracy of chromosome segregation depends on spindle driving force and the spatiotemporal regulation of spindle assembly checkpoint (SAC) on kinetochores. RPB1 is the largest subunit of RNA polymerase II, its C-terminal domain (CTD）phosphorylation on Ser2, Ser5 and Ser7 regulates transcription initiation, elongation and termination. Transcription activity is silenced during cell division, however the existing form and potential function of RPB1 is still unknown. In previous work we found RPB1 CTD phosphorylation still occured during mouse oocyte meiosis, RPB1 phosphorylated on different residues were respectively concentrated on microtubule organizing center (MTOC), spindle and kinetochores, suggesting that the RPB1 might regulate chromosome segregation through participating in the formation of meiotic spindle and the functional setup of SAC. The project intends to modify the phosphorylation patterns of RPB1 on Ser2, Ser5 and Ser7, respectively, by way of molecular mutations, thus explore the specific roles and molecular mechanisms of different phosphorylation patterns in ensuring the accuracy of chromosome segregation in oocytes, the research results will be of great significance for understanding the formation of embryo aneuploid.

卵母细胞减数分裂中染色体分离错误会导致产生非整倍体胚胎，是不育、自发性流产和出生缺陷的重要原因。染色体分离的精确性取决于纺锤体的牵引和动粒上纺锤体组装检验点的调控。RPB1是RNA聚合酶 II的最大亚基，其羧基端在Ser2、Ser5和Ser7等位点的磷酸化调节转录的起始、延伸和终止。细胞分裂期间转录静止，但RPB1的存在形式和潜在功能尚未知。前期发现小鼠卵母细胞减数分裂过程中RPB1的磷酸化仍然发生，不同位点磷酸化的RPB1分别聚集在微管组织中心、纺锤体和动粒上，提示RPB1可能通过参与纺锤体形成和纺锤体组装检验点的功能建立从而调控染色体分离。本项目拟继续通过分子突变等方式分别改变RPB1在Ser2、Ser5和Ser7的磷酸化模式，研究上述位点的磷酸化修饰在确保卵母细胞染色体精确分离中的具体作用和分子机制，研究结果将对于认识非整倍体胚胎的形成具有重要意义。

卵母细胞减数分裂过程中染色体分离错误会导致产生非整倍体胚胎，是不育、自发性流产和出生缺陷的重要原因。染色体分离的精确性取决于纺锤体的牵引和动粒上纺锤体组装检验点的调控。RPB1是RNA聚合酶 II的最大亚基，其羧基端在Ser2、Ser5和Ser7等位点的磷酸化调节转录的起始、延伸和终止。减数分裂进程中全基因组处于转录静默状态，RPB1的CTD磷酸化在减数分裂恢复后重新出现并呈现特殊的亚细胞定位方式，与纺锤体形成和SAC功能建立相关。通过分子突变技术分别改变RPB1在Ser2、Ser5和Ser7的磷酸化模式，研究上述位点的磷酸化修饰在确保卵母细胞染色体精确分离中的作用和分子机制，进一步通过显微注射RPB1 morpholino的方法敲降小鼠卵母细胞内RPB1的蛋白水平，发现卵母细胞减数分裂重启被阻滞，染色质未发生凝集，形成很多泡状结构，同时利用激酶抑制剂来特异性抑制磷酸化蛋白的表达，观察到卵母细胞减数分裂进程受到影响。以此课题为依托，我们发现PLD2参与调节小鼠卵母细胞减数分裂中微管稳定性及纺锤体趋皮质区迁移；抑制NQO2能够缓解ROS对小鼠卵母细胞减数分裂成熟和胚胎发育的影响；Stau2在小鼠卵母细胞纺锤体组装、微管与动粒连接建立及胞质分裂中发挥重要作用；Pak1活性可以通过调节H3Ser10的磷酸化而调控染色质的凝集。系列研究成果揭示了卵母细胞染色体分离的分子调控机制，为认识非整倍体胚胎的形成提供新的证据支持，同时有望为不孕不育症等相关疾病提供新的治疗靶点。