Mitochondria-anchored protein complexes in piRNA biogenesis and function

项目摘要 粗线期piRNA是哺乳动物特有的小调节RNA群体， 调节精子发生和生育能力。在小鼠中，粗线期piRNA产生自 长皮尔纳前体通过皮尔纳加工机制切割， 加载到两种细胞质PIWI蛋白MIWI和MILI上。然而， 哪些PIWI蛋白被募集到皮尔纳加工机器中以参与皮尔纳加工， 生物起源仍然难以捉摸。在这里，我们提供了初步数据，揭示了一种新的遗传 分离MIWI和MILI以差异性地进入皮尔纳加工机器。 这种分离是由一种锚定蛋白复合物介导的， 特异性地与MIWI相互作用，但不与MILI相互作用。我们假设不同的线粒体- 锚定复合物差异性地将不同PIWI蛋白引导到皮尔纳途径中 在粗线期皮尔纳加工期间。为了验证这一假设，我们将使用生物化学 和小鼠遗传学方法：1）了解特定的功能重要性， 指导皮尔纳生物发生和生殖细胞的基于DNA的蛋白质相互作用 功能; 2）定义PIWI蛋白募集和下游的偶联机制 皮尔纳加工; 3）探索特定的MILI招募机制以进入 皮尔纳途径。这些研究将提供新的见解的组织原则， 皮尔纳加工机制和对潜在机制的理解 粗线期皮尔纳生物发生及其与正常精子发生和生育力的相关性。

PROJECT SUMMARY Pachytene piRNAs are a population of small regulatory RNAs unique to mammals that regulates spermatogenesis and fertility. In mice, pachytene piRNAs are generated from long piRNA precursors through cleavage by the piRNA processing machinery and loaded onto two cytoplasmic PIWI proteins MIWI and MILI. However, the mechanism by which PIWI proteins are recruited to piRNA processing machinery to participate in piRNA biogenesis remains elusive. Here we provide preliminary data that reveal a novel genetic separation of MIWI and MILI to differentially enter into the piRNA processing machinery. This segregation is mediated by a mitochondria-anchored protein complex that specifically interacts with MIWI but not MILI. We hypothesize that distinct mitochondria- anchored complexes differentially direct different PIWI proteins into the piRNA pathway during pachytene piRNA processing. To test this hypothesis, we will use biochemical and mouse genetic approaches to: 1) understand the functional importance of specific mitochondria-based protein interactions in directing piRNA biogenesis and germ cell function; 2) define the coupling mechanism for PIWI protein recruitment and downstream piRNA processing; 3) explore a specific MILI recruiting mechanism to enter into the piRNA pathway. These studies will provide novel insight into the organizing principle of the piRNA processing machinery and the understanding of mechanism underlying pachytene piRNA biogenesis and its relevance to normal spermatogenesis and fertility.