Dissect the piRNA regulatory mechanism during spermatogenesis

项目摘要 PIWI蛋白及其相关的小RNA（piRNA）对于哺乳动物的雄性生育力是必需的。我漫长 长期目标是了解piRNA的生物学及其在男性生育中的功能。未知的机制 确定一些RNA被加载到PIWI中并成为piRNA，而另一些则不是。在这份协议中，我 建议研究决定皮尔纳形成的结构和机制因素。我假设 （1）产生piRNA的基因座的独特结构和（2）特异性转录后加工事件 靶向转录物以产生皮尔纳。然而，两者都没有得到很好的理解。在K99阶段，我将 研究皮尔纳启动子、皮尔纳前体和皮尔纳加工中间体。为了这些研究，我会 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，重现皮尔纳的产生，用于生化研究。在初步研究中，我 我已经定义了皮尔纳产生基因座的转录单位，允许它们与其他基因进行比较， 已经观察到可能负责piRNA的5 ′末端形成的片段化事件，可能偶联 PIWI装载。在本建议书的R00阶段，我将确定必要的元素和事件 并足以产生皮尔纳，并在以下背景下剖析piRNA代谢的步骤和位置： 精子发生定义皮尔纳的拟议研究将有助于我们理解为什么皮尔纳突变小鼠 不育，从而更好地了解人类男性不育症。

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.