Functional characterization of piRNPs

描述(申请人提供)：随着核糖核蛋白(RNP)复合体的发现，我们对基因表达调控的理解发生了根本性的变化。这些复合体的核心是由两个亚类组成的ArgAerte蛋白质家族的成员：AGO和Piwi。AGO蛋白与microRNAs结合。Piwi蛋白与piwi相互作用的RNA(PiRNAs)结合。Piwi蛋白是生殖系发育和生殖细胞规范所必需的。然而，许多piRNAs和Piwi蛋白的功能和RNA靶标在很大程度上是未知的。对黑腹果蝇的遗传学研究已经确定了母体基因，其产物在被称为生殖质的结构中组装，这是生殖细胞规范所必需的。这些因子包括PiRNA结合蛋白Aubergine、精氨酸蛋白甲基转移酶(PRMT5)及其辅助因子、都铎蛋白和Vasa RNA解旋酶。虽然在果蝇中已经很好地描述了生殖质组装的遗传途径，但对生殖质蛋白的关系和功能还不是很清楚。种质还含有被认为是生殖细胞规范所必需的RNA，但其身份大多尚不清楚。果蝇生殖系发育和生殖细胞规范所需的大多数基因在其他动物(如小鼠)的生殖系中具有进化保守的作用。我们的研究正在揭示关键种质成分的重要关系和功能。我们发现，由PRMT5催化的Piwi蛋白的精氨酸甲基化是一种进化保守的修饰，介导了与Tudor蛋白的相互作用。我们还发现Vasa及其小鼠同源物含有甲基化的精氨酸。在目标1中，我们将从功能上表征果蝇体内的piRNP复合体及其RNA靶标。在目标2中，我们将从功能上表征小鼠肌肉中的piRNP复合体及其RNA靶标。对这两种生物的研究将突出piRNP功能和生殖细胞规范和发育的保守角色和途径，但也可能揭示物种特有的功能。这项建议中详细介绍的研究将涉及piRNPs和相关蛋白质及其RNA靶标功能的基本方面，以及这些因素在生殖细胞规格和生殖系发育中的作用。 公共卫生相关性： 我们将在生殖细胞规范中研究由小RNA控制的基因表达调控。反过来，这些知识将导致对理解干细胞生物学和影响生殖和发育的疾病的重要见解。

DESCRIPTION (provided by applicant): Our understanding of gene expression regulation has changed radically with the discovery of ribonucleoprotein (RNP) complexes programmed with small RNAs. At the core of these complexes are members of the Argonaute family of proteins that is comprised of two subclades: Ago and Piwi. Ago proteins bind to microRNAs. Piwi proteins bind to piwi-interacting RNAs (piRNAs). Piwi proteins are essential for germline development and germ cell specification. However, the function and RNA targets for many piRNAs and Piwi proteins are largely unknown. Genetic studies in Drosophila melanogaster have identified maternal genes whose products assemble in structures known as germ plasm, and which are required for germ cell specification. Among these factors are the piRNA binding protein Aubergine, an arginine protein methyltransferase (PRMT5) and its cofactor, the Tudor protein and the Vasa RNA helicase. Although the genetic pathway of germ plasm assembly has been well characterized in Drosophila, the relationship and functions of germ plasm proteins is not well understood. Germ plasm also contains RNAs, that are thought to be essential for germ cell specification but whose identities are mostly unknown. Most of the genes that are required for germline development and germ cell specification in Drosophila have evolutionary conserved roles in the germline of other animals, such as mice. Our studies are uncovering important relationships and functions of critical germ plasm components. We find that arginine methylation of Piwi proteins, catalyzed by PRMT5, is an evolutionary conserved modification that mediates interaction with Tudor proteins. We also find that Vasa and its mouse homolog, contain methylated arginines. In Aim 1, we will functionally characterize piRNP complexes and their RNA targets in Drosophila melanogaster. In Aim 2, we will functionally characterize piRNP complexes and their RNA targets in Mus musculus. Studying both organisms will highlight conserved roles and pathways for piRNP function and germ cell specification and development, but will also likely uncover species-specific functions. The studies detailed in this proposal will address fundamental aspects of the function of piRNPs and associated proteins and their RNA targets, and the role of these factors in germ cell specification and germline development. PUBLIC HEALTH RELEVANCE: We will investigate gene expression regulation controlled by small RNAs in specification of germ stem cells. In turn, this knowledge will lead to important insights towards understanding stem cell biology and diseases that affect reproduction and development.