MARF1调控卵子减数分裂和反转座子沉默的作用和机制

Production of normal eggs and sperm require completion of meiosis, for segregation of homologous chromosomes, and silencing of retrotransposons, for defence of genomic integrity.Precise control of these processes is critical for the success of reproduction, and abnormalities in any of these can lead to infertility, miscarriage or birth defects, and endanger future generations. A group of proteins (e.g. tudor domain containing proteins and PIWI-family members) and their interacting partners (e.g. piRNAs), which localize to a germ cell-specific aggregates of ribonucloprotein (RNP) complexes commonly known as nuage, are found to be essential for the control of both processes in a variety of species. However, in mammals, mutations of genes encoding these proteins and abrogation of piRNA production have been reported to only affect male germ cell development and function,but not that of females. Excitingly, we have identified recently a novel Riken gene that functions in the control of both meiotic progression and retrotransposon silencing in mouse oocytes. Mutations of this gene cause female only infertile phenotype owing to meiotic arrest in oocytes and ovulation of immature germinal vesicle (GV)stage oocytes, we therefore named it Marf1 (meiosis arrest female 1). The function of Marf1 orthologs and/or homologs in any other model organisms has not yet been appreciated. We found that Marf1 is highly expressed by oocytes and its mutation causes up-regulation of a cohort of transcripts, of which up-regulation of protein phosphatase 2 catalytic subunit B (PPP2CB) is key to the meiotic arrest phenotype. This is consistent with the presence of a predicted RNase-like domain in MARF1 protein. Moreover, Iap and Line1 retrotransposon mRNAs are also up-regulated, and, concomitantly, DNA double-strand breaks are elevated in mutant oocytes. Therefore, MARF1 is indispensible for control of meiotic progression, retrotransposon repression, and transcriptome steady state in mouse oocytes. MARF1, by suppressing levels of specific transcripts, is a "master" regulator of key oogenic processes leading to female fertility and the development of healthy offspring. However, the exact mechanisms by which MARF1 control these essential oogenic processes remain unclear, hence become the main aim of this project proposal. In this project we aim to specifically address the following questions: (1) Does MARF1 have RNase-slicer activity? (2) What are the specific RNA and protein binding partners of MARF1? (3) Does MARF1 interact with or participate in piRNA production in oocyes? We expect that, by finding the answers to these questions, we will be able to understand the molecular mechanisms by which MARF1 control meiosis and retrotransposon silencing in mammalian cytes, which will eventually provide new insights into the control of mammalian oocyte development, and help to define the molecular determinants of egg quality.

完成减数分裂和维持反转座子沉默是产生正常卵子进而保证生殖过程成功的关键；其中任何差错都能导致不育、自发流产、出生缺陷甚至危及后代。虽然调控这两个环节的许多蛋白（像TDRDs和PIWI家庭成员）和piRNAs相继在雄性哺乳动物生殖细胞被发现，但所有这些分子都不影响雌性哺乳动物的生育。我们最近发现了一个能够同时调控小鼠卵母细胞减数分裂和反转座子沉默的全新Riken基因。该基因主要在卵子表达，发生突变时卵母细胞不能恢复减数分裂、雌鼠排出GV-期未成熟卵子，进而不育。然而雄性突变鼠则正常。我们命名该基因为Marf1。Marf1在其他模式生物的同源基因的功能迄今未知。MARF1蛋白拥有一个"RNase-样"和几个同时也存在于果蝇Oskar蛋白和小鼠TDRD5,7蛋白的"OST-HTH 或LOTUS"结构域。本项目在我们近期工作基础上将对MARF1调控卵母细胞的减数分裂和反转座子沉默机制展开细致研究。

卵母细胞成熟缺陷 （包括不能正常恢复减数分裂、减数分裂进程缺陷以及染色体分离异常等）是造成卵子质量低下进而导致女性不孕不育以及出生缺陷的重要因素之一。本项目在前期发现调控卵母细胞减数分裂和遗传信息稳定传递的全新蛋白MARF1的基础上，按照原计划任务书的既定研究方案，系统地研究了MARF1蛋白对卵母细胞成熟以及基因组完整性维持的调控作用及机制。揭示了MARF1在卵泡不同发育时期的卵母细胞内的表达变化规律，以及该变化与MARF1发挥抑制卵母细胞反转座子沉默作用时间的关联性。通过多次尝试，最终成功制备了可用于免疫荧光和免疫共沉淀试验的小鼠MARF1特异性抗体，利用该抗体揭示了MARF1 在卵母细胞成熟过程中的表达变化及细胞内定位。发现MARF1与减数分裂纺锤体和动粒共定位，并且调控染色体排列以及精密分离，进而维持基因组的完整性和遗传信息的稳定传递。MARF1通过调控染色质重塑、转录沉默以及组蛋白的修饰来控制卵母细胞的表观遗传成熟过程，为研究卵母细胞成熟调控的分子机制开拓了新思路。通过与复旦大学麻锦标教授合作，我们成功解析了MARF1蛋白关键结构域“NYN”以及“LOTUS”的晶体结构，并通过定点突变“NYN”结构域中对于其酶的活性起决定作用的保守氨基酸位点证明了其在卵母细胞内具有特异的RNAse活性。在此基础上，利用CRISPER-CAS9基因编辑技术成功制备了该保守氨基酸位点突变的敲入小鼠，为进一步利用遗传学手段研究该具有RNAse活性的“NYN”结构域的在体功能提供了切实的保障。综上，该项目揭示了MARF1通过参与维持卵母细胞内mRNA的稳态来调控卵母细胞的核成熟、质成熟、表观遗传成熟和基因组的完整性，这不仅为深入探讨决定卵母细胞质量的分子基础奠定了坚实的基础，而且对于临床上因卵母细胞质量低下所致的不孕不育的诊疗和出生缺陷的防范具有重要指导意义。