The Function of piRNAs and the piRNA Complex

小的非编码RNA是生物体基因调控回路的核心组成部分。为了正常的健康和发育，生物体利用含有内源性小RNA的蛋白质复合物通过沉默机制控制成千上万的其他基因。模式生物中的遗传学暗示了小RNA、Argonaute蛋白和调节染色质结构的过程之间的联系，表明RNA干扰（RNAi）途径可以影响基因组的大结构域。我们在哺乳动物中寻找类似的联系，指导我们从大鼠睾丸中纯化皮尔纳复合物（piRC），该复合物至少由哺乳动物Piwi和Piwi相互作用RNA组成。fiwi相互作用RNA（piRNA）是比microRNA长的不同种类的小RNA，并且来源于1- 100 kb长的基因间簇。Piwi是Argonaute家族的一个分支，对苍蝇和雄性小鼠的生育力至关重要，并且在遗传上与转座因子的沉默有关。尽管有这些特征，但仍然缺乏对piRC分子功能的严格理解。 该提案旨在通过这些特定目标来剖析piRNA和piRC的分子功能：1）皮尔纳复合物的功能分析; 2）皮尔纳簇转录的调控; 3）理解piRNA的起源。我们将应用我们目前的生物化学技术来探测piRC的功能活性，并开发遗传和细胞生物学方法来了解皮尔纳簇的作用。Piwi蛋白已被证明对动物（包括哺乳动物）的生殖细胞发育至关重要。然而，这种核糖核蛋白的机制理解和基因调控潜力仍然是未知的。该研究项目旨在开发系统，推动与儿童健康和人类发展相关的主题取得进展。

Small noncoding RNAs are central components ofthe gene regulatory circuit in organisms. For proper health and development, organisms employ protein complexes containing small endogenous RNAs to control thousands of other genes through silencing mechanism. Genetics in model organisms implicate a link between small RNAs, Argonaute proteins and processes that regulate chromatin structure, suggesting that RNA interference ^RNAi) pathways can impact large domains of the genome. Our search for an analogous link in mammals directed us to purify from rat testes the piRNA Complex (piRC), which consists at least of mammalian Piwi and the Piwi-interacting RNAs. Fiwi-interacting RNAs (piRNAs) are a diverse class of small RNAs that are longer than microRNAs and derive from intergenic clusters of between 1-lOOkb long. Piwi, a subclade of the Argonaute family, is essential for fertility in flies and male mice, and has been genetically implicated in silencing of transposable elements. Despite these features, a rigorous understanding of piRC's molecular function is still lacking. This proposal seeks to dissect the molecular function ofthe piRNAs and piRC through these specific aims: 1) Functional analysis ofthe piRNA Complex; 2) Regulation of piRNA cluster transcription; 3) Understanding the Genesis of piRNAs. We will apply our current biochemical techniques to probe piRC's functional activity, and develop genetic and cell biology methodologies to understand the role of piRNA clusters. Piwi proteins have been shown to be essential for proper germ cell development in animals, including mammals. However, the mechanistic understanding and gene regulatory potential ofthis ribonucleoprotein is still uncharted. This research project aims to develop systems that will drive progress in a subject relevant to child health and human development.