Function of PIW1/Argonuate Proteins in Spermatogenesis

描述（申请人提供）：生物医学研究中最令人兴奋的进展之一是发现了小RNA介导的基因调控机制。自2002年以来，R 01支持的我们的研究为这一进步做出了重大贡献：在上一个资助期，我们发现并表征了哺乳动物Piwi/Argonaute蛋白的功能，这些蛋白是小RNA机制的核心。在当前的资助期开始时，我们进一步发现Piwi蛋白结合到一个复杂的类别，即超过60，000个小的非编码RNA，我们称之为PIWI相互作用RNA（piRNA）。大多数piRNA由基因间“垃圾”DNA编码，其主要是异染色质。piRNA的发现揭示了生物学的一个新的和令人惊讶的复杂维度，并被《科学》杂志评为2006年十大突破之一。值得注意的是，Piwi蛋白和piRNA仅在生精细胞中大量表达。我们和其他人已经证明Piwi蛋白在果蝇、C.线虫、斑马鱼和小鼠。我们还发现，人类piwi基因（hiwi）的过度表达与乳腺癌高度相关。为了探索Piwi蛋白的生精功能的机制，我们最近在果蝇中证明了Piwi蛋白调节表观遗传状态和转座子活性，并且在小鼠中证明了MILI也与染色质结合。尽管取得了这一令人兴奋的进展，但piRNA的功能尚未在任何系统中得到直接证实。该提案的目标是证明piRNA在精子发生过程中的功能。我们的工作假设是piRNA引导核PIWI蛋白到特定的基因组位点进行表观遗传调节，在转座子的情况下，这导致它们的沉默。为了验证这一假设， 为了探索piRNA的功能，我们提出：（1）确定皮尔纳簇在精子发生过程中的生物学功能。(2)研究特定piRNA对基因组的表观遗传效应。(3)确定PIWI靶向基因组序列是否需要piRNA。(4)确定抑制转座是否需要piRNA。

DESCRIPTION (provided by applicant): One of the most exciting advances in biomedical research is the discovery of small RNA- mediated gene regulation mechanisms. Our research supported by this R01 since 2002 has contributed significantly to this advancement: In the previous funding period, we discovered and characterized the function of mammalian Piwi/Argonaute proteins that are central to small RNA mechanisms. In the beginning of the current funding period, we further discovered that Piwi proteins bind to a complex class of more than 60,000 small non-coding RNAs that we called PIWI-interacting RNAs (piRNAs). Most piRNAs are encoded by intergenic "junk" DNA that is mostly heterochromatin. The discovery of piRNAs reveals a new and surprisingly complex dimension of biology, and was voted by the Science magazine as one of the ten Breakthroughs of 2006. Remarkably, Piwi proteins and piRNAs are abundantly expressed only in spermatogenic cells. We and others have shown that Piwi proteins play key roles in spermatogenesis in Drosophila, C. elegans, zebrafish, and mice. We have also shown that the overexpression of the human piwi gene (hiwi) is highly correlated to seminomas. To explore the mechanisms underlying the spermatogenic functions of Piwi proteins, we have recently demonstrated in Drosophila that Piwi proteins regulate the epigenetic state and transposon activity and in mice that MILI also binds to chromatin. Despite this exciting progress, the function of piRNAs has not been directly demonstrated in any system. The goal of this proposal is to demonstrate the function of piRNAs during spermatogenesis. Our working hypothesis is that piRNAs guide nuclear PIWI proteins to specific genomic loci for epigenetic regulation, which, in the case of transposons, lead to their silencing. To test this hypothesis and to explore the function of piRNAs, we propose to: (1) Determine the biological function of piRNA clusters during spermatogenesis. (2) Investigate the epigenetic effect of specific piRNAs to the genome. (3) Determine whether piRNAs are required for PIWI-targeting to genomic sequences. (4) Determine whether piRNAs are required for suppressing transposition.