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电路的中心组件。为了正常的健康和发育，生物体利用含有内源小RNA的蛋白质复合体，通过沉默机制来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、ArgAerte蛋白质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范围区域。 这位候选人在哺乳动物中寻找类似的联系，这让他从大鼠睾丸中纯化了piRNA复合体(Pirc)，它由哺乳动物Piwi、与Piwi相互作用的RNA和RecQ1 DNA解旋酶组成。Piwi相互作用RNA(PiRNAs)是一类比microRNAs更长的小RNA，来自1-100kb的基因间簇。Piwi是ArgAerte家族的一个亚类，是男性生育所必需的，而RecQ1与一种具有基因沉默作用的脉孢子菌基因有关。尽管有这些特点，但对Pirc的分子功能仍缺乏严格的了解。 本申请旨在通过以下特定目的来剖析piRNAs和Pirc的分子功能：1)体外分析piRNA复合体的功能；2)描述piRNA簇染色质的功能和调节；以及3)了解piRNAs的起源。这位候选人将应用他目前的生化技术来探索Pirc的功能活性，并开发遗传和细胞生物学方法来了解piRNA簇的作用。这项申请寻求支持，以促进对动物RNAi这一新机制的研究，并描述了一个综合计划，该计划将发展候选人的职业生涯，并向学术机构的独立研究职位过渡。深入了解Pirc的功能将有助于深入了解影响人类发育和生育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.