母源性RNF114蛋白在合子基因组激活过程中的作用及机制研究

A large number of maternal factors accumulated during the process of oocyte maturation, these maternal factors play an important role in the subsequent transition of the oocyte into the embryo. Zygotic genome activation (ZGA) is the key events during the oocyte-to- embryo transition, its maternal regulation mechanism is a more concerned, but still not clarified scientific issue. RNF114 is one of the proteins predominantly expressed in mouse oocytes, which was identified by our laboratory. Under the support of National Natural Science Foundation of China (Completed project, 31171113), we have proved that RNF114 had E3 ubiquitin ligase activity, screening and confirming TAB1 to be its substrate in the early embryo, the results of siRNA microinjection experiments indicate that RNF114 play a role in the developmental stage from 2 to 4 cells by degrading TAB1, ensure the early embryo developing beyond 2-cell stage. Recently, we used TALEN technology to build Rnf114 knockout mice, and found Rnf114 -/- female mice was infertile, the maternal RNF114 deficient embryo got by mating Rnf114 -/- female mice with wild-type males entirely arrested at 2-cell stage, which is consistent with the results of in vitro experiment. In mouse, massive ZGA happened at the 2-cell stage, the preliminary result of EU incorporation experiments prompted ZGA defect in the RNF114 knockdown embryos . Therefore, this project intends to use Rnf114 knockout mice, in-depth study the role of maternal RNF114 protein in ZGA, and illustrate its regulating mechanisms on ZGA by degradating its substrate proteins and / or non-degradation pathways, and utilizing immature oocyte to explore its role in human early embryonic development process. The study will be helpful to understand the mechanism of maternal regulation of ZGA, find the causes of clinical embryo development block and provide a scientific basis for clinical diagnosis and treatment.

卵母细胞成熟过程中累积的母源因子在后续母胚转换中发挥重要作用。合子基因组激活（ZGA）是母胚转换的关键事件，其母源调控是备受关注的科学问题。RNF114是本实验室筛选到的一个卵细胞优势表达蛋白，在已结题项目的资助下，我们证明该蛋白具有E3连接酶活性，筛选到TAB1为其在早期胚胎中的底物，siRNA注射实验表明其可通过降解TAB1在2至4细胞发育阶段发挥作用。近期、我们构建了Rnf114敲除小鼠，发现母源RNF114缺失的胚胎完全阻滞于2细胞期；2细胞期正是小鼠大规模ZGA发生的时期，EU掺入实验提示RNF114敲低胚胎中ZGA发生障碍。因此，本项目拟利用敲除小鼠，阐明母源RNF114蛋白在ZGA中的作用，揭示其通过降解底物和/或非降解途径调控ZGA的机制，并利用未成熟卵探讨其在人早期胚胎发育过程的作用。该研究将帮助理解ZGA的母源调控机制，并为理解临床早期胚胎发育阻滞的原因及诊疗提供依据。

RNF114是本实验室前期筛选到的卵母细胞优势表达蛋白，在前一个自然科学基金（31171113）的资助下，我们证明了RNF114蛋白具有E3连接酶活性，并利用体外实验证实RNF114通过调控下游底物蛋白TAB1在母-胚转换过程中发挥重要作用。随后、我们构建了Rnf114敲除小鼠，初步发现母源RNF114缺失的胚胎出现明显的发育障碍，与体外实验结果一致。在此基础上本项目着重研究了母源RNF114蛋白在母-胚转换过程中的具体作用及机制，研究表明Rnf114-/-小鼠的卵母细胞发育、排卵及受精均无明显异常，而母源RNF114缺失的胚胎完全阻滞于2细胞阶段，EU掺入实验、标志分子的免疫荧光染色及大规模转录组测序结果均提示母源RNF114缺失的胚胎中合子基因组激活（ZGA）出现严重障碍。进一步我们对其机制进行探讨，结果表明母源RNF114的缺失一方面通过影响其底物TAB1的降解抑制NF-kB通路的激活，从而影响转录活化；另一方面，RNF114也可能通过调节其它底物蛋白参与母-胚转换和ZGA过程，通过对Rnf114-/-小鼠的卵母细胞进行蛋白质组检测，我们关注到CBX5蛋白，进一步研究结果证明RNF114调控CBX5的泛素化降解，在母源RNF114缺失的2细胞胚胎中，CBX5异常高表达，而CBX5在早期胚胎中的过表达可导致胚胎发育自2细胞阶段发生阻滞，ZGA出现障碍。本研究证实了RNF114蛋白的母源调控效应，发现了其通过调控底物蛋白在major ZGA中发挥重要作用。ZGA是母-胚转换的关键事件，对于早期胚胎发育具有至关重要的意义，本研究有助于我们深入理解母源性因子对ZGA的调控作用这一重要的生殖生物学问题。近期，我们在临床胚胎发育障碍患者中检测到 RNF114的遗传变异，后续研究的进一步深入也将有助于对人早期胚胎发育机制的理解，并有望为临床早期胚胎发育障碍的诊治提供新的线索和可能途径。