PIWI/piRNA代谢调控在哺乳动物精子形成中的功能机制研究

PIWI proteins that complex with piRNAs, a class of animal germ-cell specific small non-coding RNA, protect the stability and integrity of germ-cell genome by preventing the activity of mobile genetic elements, and is essencial to gametogenesis in animal. Recent studies have advanced our understanding of the biogenesis and function of PIWI/piRNA complex. Most recently, we explored the metabolism of PIWI/piRNA complex. Our data showed that piRNAs trigger MIWI ubiquitination and removal by APC/C-ubiquitin pathway in late spermatogenesis. Moreover, MIWI degradation in turn leads to piRNA elimination, suggesting a feed-forward mechanism for coordinated removal of the MIWI/piRNA complex at a specific developmental stage. This work reveals the molecular mechanism of PIWI/piRNA metabolism; however, whether and how the proper turnover of PIWI/piRNA impacts spermiogenesis remains largely unknown. Our preliminary results showed that inhibition of MIWI degradation by expressing mutant form of MIWI that is resistant to APC/C-mediated ubiquitination prevented formation of mature sperms. More interestingly, we identified three D-box mutations of Hiwi gene from asthenozoospermia/azoospermia patients. These results imply the necessity of proper turnover of PIWI to normal spermatogenesis in mammal. In this research plan, we propose two aims. First, we will further investigate the correlation between ubiquitination-resistant Hiwi mutations and male sterility. Second, we will use transgenic mouse models that express Piwi D-box mutants and study the molecular mechanisms of male sterility caused by ubiquination-deficient PIWI mutants. Data generated from this project will reveal the role and mechanism of proper temporal regulation of the PIWI/piRNA metabolism in mammalian spermiogenesis, bring new insights into molecular information for asthenozoospermia/azoospermia, and provide molecular targets for diagnosis and treatment of male sterility.

PIWI蛋白与动物生殖细胞特异性piRNA结合，沉默基因组移动性遗传元件，维持生殖细胞基因组稳定性和完整性，为配子形成必需。近期的研究已揭示PIWI/piRNA的作用机制，而我们新近对PIWI/piRNA的代谢调控进行探索，发现小鼠piRNA在精子形成后期触发其结合蛋白PIWI（MIWI）经APC/C泛素化降解，揭示PIWI与piRNA以协同模式在后期精子细胞中共同清除的新机制，但目前尚不确定该代谢调控是否具有重要生物学意义。初步的研究发现，在小鼠精子细胞中稳定MIWI将阻断精子成熟，在少弱/无精症患者Piwi基因（Hiwi）存在拮抗泛素化修饰突变。在本申请项目中，我们将利用转基因小鼠模型，深入研究PIWI泛素化修饰缺陷致雄性/男性不育的分子机制。项目研究获得结果将揭示PIWI/piRNA代谢调控在哺乳动物精子形成中的功能机制，可为精子生成障碍提供分子信息，并为男性不育症诊治提供分子靶。

研究表明，Piwi/piRNA通路是动物生殖细胞特异性的小RNA通路。piRNA通路拮抗基因组转座遗传元件、维持生殖细胞基因组稳定性和完整性，对动物生殖细胞发育及个体生殖至关重要，但目前对该通路缺陷与男性不育症之间的联系还知之甚少，对该通路是否具有沉默转座元件以外的功能也不甚了解。在该项目支持下，我们探索了HIWI（人PIWI）代谢异常在无精症发生中的功能机制，并研究了小鼠piRNA及其结合蛋白MIWI（小鼠PIWI）对睾丸组织mRNA稳定性的影响，主要获得了以下研究结果：1）从无精患者中鉴定了拮抗HIWI（人源PIWI）蛋白泛素化修饰的D-box突变，并通过Knock-in小鼠模型发现，此类突变阻碍精子变形过程中的组蛋白-鱼精蛋白交换并导致精子形成异常及雄性不育；2）发现小鼠piRNA可指导MIWI利用其slicer活性对靶mRNA直接切割降解的新功能，并证明这一机制对小鼠精子形成至关重要。此外，项目组还研究了miRNAs在肿瘤发生发展及肿瘤细胞代谢等过程中的功能机制。共发表了项目资助标注论文3篇(Cell, accepted; Cell Res, 2014; EMBO J, 2015)。此外，在项目执行期间，培养了2名博士研究生毕业取得博士学位，1名硕士研究生毕业取得硕士学位。