Dissect the piRNA regulatory mechanism during spermatogenesis

项目总结： Piwi蛋白及其相关的小RNA(PiRNAs)是哺乳动物雄性生殖所必需的。我的龙 学期目标是了解piRNAs的生物学及其在男性生育中的作用。未知机制 确定某些RNA已加载到PIWI中并成为piRNAs，而另一些则不是。在这笔赠款中，我 建议研究决定piRNA形成的结构和机制因素。我假设 (1)piRNA产生基因座的独特结构和(2)特定的转录后加工事件 将转录本作为生产piRNA的靶标。然而，这两种情况都没有得到很好的理解。在此提案的K99阶段，我将 研究piRNA启动子、piRNA前体和piRNA加工中间体。为了这些研究，我将 开发深度测序和荧光原位杂交的新策略。我还建议建立一个 无细胞系统，为生化研究概括piRNA的生产。在初步研究中，我有 已经定义了piRNA产生基因座的转录单位，允许它们与其他基因进行比较，并且我 我观察到了可能导致5个ʹ末端形成piRNA的碎片事件，可能是偶联 加装了PIWI。在本提案的R00阶段，我将确定必要的元素和事件 并且足以产生piRNA，并在以下背景下剖析了piRNA代谢的步骤和位置 精子发生。拟议中的定义piRNA的研究将有助于我们理解为什么piRNA突变小鼠 都是不育的，这让人们对人类男性不育有了更好的认识。

Project Summary  PIWI proteins and their associated small RNAs (piRNAs) are essential for male fertility in mammals. My long term goal is to understand the biology of piRNAs and their function in male fertility. Unknown mechanisms determine that some RNAs are loaded into PIWI and become piRNAs, while others are not. In this grant, I propose to study the structural and mechanistic factors that determine piRNA formation. I hypothesize that both (1) the unique structure of piRNA-producing loci and (2) specific post-transcriptional processing events target a transcript for piRNA production. Yet, neither is well understood. In the K99 phase of this proposal, I will study piRNA promoters, piRNA precursors, and piRNA processing intermediates. For these studies, I will develop new strategies for deep sequencing and fluorescent in-situ hybridization.I also propose to set up a cell-free system that recapitulates piRNA production for biochemical studies. In preliminary studies, I have already defined the transcription unit of piRNA producing loci, allowing their comparison with other genes, and I have observed a fragmentation event probably responsible for 5ʹ′ end formation of piRNAs, perhaps coupling with PIWI loading. In the R00 phase of this proposal, I will identify the elements and events that are necessary and sufficient for piRNA production, and dissect the steps and locations of piRNAs metabolism in the context of spermatogenesis. The proposed study to define a piRNA will help us to understand why piRNA mutant mice are infertile, leading to a better understanding of human male infertility.