Function of PIWI/Argonuate Proteins in Spermatogenesis

描述(申请人提供)：Piwi/ArgAerte(AGO)是唯一已知的具有干细胞自我更新功能的蛋白质家族，在动植物界都高度保守。这些蛋白还在生殖系发育和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蛋白调节精子发生的分子机制。这也将有助于理解干细胞分裂和由这一蛋白质家族介导的其他发育过程。我们的策略是以果蝇为模型来研究这些蛋白质在配子发生中的作用，然后将我们从果蝇那里学到的东西推广到哺乳动物和临床环境中。特别是，在目前的资助时期，我们已经证明了mii是基因家族中的一个小鼠成员，对于精原干细胞的分裂和减数分裂是必不可少的，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基因家族的头两次活体研究。此外，我们已经证明，人类piwi基因的过度表达与睾丸精原细胞瘤高度相关。我们在这里的具体目的是研究mii和miwi调控精子发生的分子机制。已知Piwi/AGO家族蛋白通过RNAi、miRNA和表观遗传途径负向调节基因表达。然而，我们最新的研究表明，miwi和mili可能通过miRNA介导的机制对靶mRNAs的翻译和稳定性进行积极的调节。这些发现为我们提供了一个无与伦比的机会，让我们发现一种新的基因调控机制，这是生殖和干细胞生物学的基础。我们的工作假说有三个组成部分：第一，Mili和Miwi结合并稳定他们的目标生精事件所需的靶mRNAs；第二，这一功能与翻译调控相耦合；第三，这一功能是由miRNAs介导的。为了验证这一假设，我们建议：(1)系统地识别Mili和Miwi的靶mRNAs。(2)研究MILI和MIWI结合对靶mRNAs稳定性的影响。(3)确定Mili和Miwi是否通过调节靶mRNAs的翻译来控制其稳定性。(4)鉴定MILI/MIWI靶向miRNAs，并确定其在mRNA调控中的功能。由于Piwi/AGO蛋白已证明在肿瘤发生中发挥作用，拟议的研究应具有重大的健康意义。

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.