The Function of piRNAs and the piRNA Complex

描述（由申请人提供）：小的非编码RNA是生物体中基因调控回路的中心组成部分。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，这表明RNA干扰（RNAi）途径可以影响基因组的大结构域。 这位候选人在哺乳动物中寻找类似的联系，指导他从大鼠睾丸中纯化皮尔纳复合物（piRC），该复合物由哺乳动物Piwi，Piwi相互作用的RNA和RecQ 1 DNA解旋酶组成。Piwi相互作用RNA（piRNA）是一类比microRNA长的不同类型的小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，对男性生育力至关重要，而RecQ 1与具有基因沉默作用的脉孢菌基因有关。尽管有这些特征，但仍然缺乏对piRC分子功能的严格理解。 本申请试图通过这些特定目的剖析piRNA和piRC的分子功能：1）皮尔纳复合物的体外功能分析; 2）描绘皮尔纳簇染色质的功能和调节;以及3）理解piRNA的起源。候选人将应用他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。该申请寻求支持，这将有助于研究动物中的这种新的RNAi机制，并描述了一个综合计划，该计划将发展候选人的职业生涯和过渡到学术机构的独立研究职位。对piRC功能的深入了解将有助于深入了解影响人类发育和生育能力的基本基因和染色质调控过程。

DESCRIPTION (provided by applicant): Small noncoding RNAs are central components of the 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 among small RNAs, Argonaute proteins, and processes that regulate chromatin structure, suggesting that RNA interference (RNAi) pathways can impact large domains of the genome. The candidate's search for an analogous link in mammals directed him to purify from rat testes the piRNA complex (piRC), which consists of mammalian Piwi, the Piwi-interacting RNAs, and a RecQ1 DNA helicase. Piwi-interacting RNAs (piRNAs) are a diverse class of small RNAs that are longer than microRNAs and derive from intergenic clusters of between 1-100 kb long. Piwi, a subclade of the Argonaute family, is essential for male fertility, while RecQ1 is related to a Neurospora gene with gene silencing roles. Despite these features, a rigorous understanding of piRC's molecular function is still lacking. This application seeks to dissect the molecular function of the piRNAs and piRC through these specific aims: 1) in vitro functional analysis of the piRNA complex; 2) delineating the function and regulation of piRNA cluster chromatin; and 3) understanding the genesis of piRNAs. The candidate will apply his current biochemical techniques to probe piRC's functional activity, and develop genetic and cell biology methodologies to understand the role of piRNA clusters. This application seeks support that will facilitate investigations of this new mechanism for RNAi in animals, and describes an integrative plan that will develop the career and transition of the candidate towards an independent research position at an academic institution. A deeper understanding of piRC's function will yield insight to fundamental gene and chromatin regulatory processes that impact human development and fertility.