Function of PIWI/Argonuate Proteins in Spermatogenesis

描述（由申请人提供）：Piwi/Argonaute （Ago）是唯一已知的在动物和植物界高度保守的干细胞自我更新功能蛋白家族。这些蛋白也在种系发育和RNAi/ mirna介导的机制中发挥关键作用。我们的长期目标是了解Piwi/Ago蛋白调控精子发生的分子机制。这也将有助于理解干细胞分裂和其他由该蛋白家族介导的发育过程。我们的策略是用果蝇作为模型来研究配子发生中的这些蛋白质，然后将我们从果蝇身上学到的东西进一步应用到哺乳动物和临床环境中。特别是，在目前的资助期内，我们已经证明了mili，一个基因家族的小鼠成员，是精原干细胞分裂和减数分裂所必需的，而miwi，另一个成员，是精子发生的关键调节因子。这是对哺乳动物系统中piwi/ago基因家族的头两项体内研究。此外，我们已经表明，人类piwi基因hiwi的过表达与睾丸精原细胞瘤高度相关。我们的具体目的是研究mili和miwi调节精子发生的分子机制。已知Piwi/Ago家族蛋白通过RNAi、miRNA和表观遗传途径负调控基因表达。然而，我们的最新研究表明，miwi和mili积极调节目标mrna的翻译和稳定性，可能是通过mirna介导的机制。这些发现为我们发现一种新的基因调控机制提供了一个无与伦比的机会，这是生殖和干细胞生物学的基础。我们的工作假设有三个组成部分：首先，Mili和Miwi结合并稳定其目标生精事件所需的目标mrna；其次，该功能与翻译调节相耦合；第三，该功能是由mirna介导的。为了验证这一假设，我们提出：(1)系统地鉴定Mili和Miwi的靶mrna。(2)表征Mili和Miwi结合对靶mrna稳定性的影响。(3)确定Mili和Miwi是否通过调控靶mrna的翻译来控制其稳定性。(4)鉴定Mili/ miwi靶点mirna，确定其调控mRNA的功能。由于Piwi/Ago蛋白已被证明在肿瘤发生中起作用，因此拟议的研究应具有重要的健康意义。

DESCRIPTION (provided by applicant): Piwi/Argonaute (Ago) is the only known protein family with function in stem cell self-renewal highly conserved in both animal and plant kingdoms. These proteins also play key roles in germline development and RNAi/miRNA-mediated mechanisms. Our long term goal is to learn molecular mechanisms mediated by the Piwi/Ago proteins in regulating spermatogenesis. This will also contribute to understanding of stem cell division and other developmental processes mediated by this family of proteins. Our strategy has been to use Drosophila as a model to study these proteins in gametogenesis, and then to further what we learn from Drosophila to mammalian and clinical settings. Particularly, in the current funding period, we have demonstrated that mili, a murine member of the gene family, is essential for spermatogonial stem cell division and meiosis, yet miwi, another member, is a key regulator of spermiogenesis. These represent the first two in vivo studies of the piwi/ago gene family in mammalian systems. Moreover, we have shown that the overexpression of a human piwi gene, hiwi, is highly correlated to testicular seminomas. Our specific aim here is to study the molecular mechanisms through which mili and miwi regulate spermatogenesis. Piwi/Ago family proteins are known to negatively regulate gene expression via RNAi, miRNA, and epigenetic pathways. Our latest studies, however, indicate that miwi and mili positively regulate translation and stability of target mRNAs, possibly via a miRNA-mediated mechanism. These findings provide an unparalleled opportunity for us to discover a novel mechanism of gene regulation that is fundamental to reproductive and stem cell biology. Our working hypothesis has three components: First, Mili and Miwi bind to and stabilize target mRNAs required for their target spermatogenic events; second, this function is coupled to translational regulation; third, this function is mediated by miRNAs. To test this hypothesis, we propose to: (1) systematically identify target mRNAs of Mili and Miwi. (2) Characterize the effect of Mili and Miwi binding on the stability of target mRNAs. (3) Determine if Mili and Miwi controls the stability of target mRNAs by regulating their translation. (4) Identify Mili/Miwi-target miRNAs and determine their function in regulating mRNA. Because Piwi/Ago proteins have demonstrated roles in oncogenesis, the proposed studies should bear significant health implications.