Functional characterization of piRNPs

描述（由申请人提供）：我们对基因表达调控的理解随着小RNA编程的核糖核蛋白（RNP）复合物的发现而发生了根本性的变化。这些复合物的核心是Argonaute蛋白质家族的成员，该家族由两个亚支组成：Ago和Piwi。Ago蛋白与microRNA结合。Piwi蛋白与Piwi相互作用RNA（piRNA）结合。Piwi蛋白是生殖细胞发育和生殖细胞特化所必需的。然而，许多piRNA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，其产物组装在称为种质的结构中，并且是生殖细胞特化所必需的。这些因子包括皮尔纳结合蛋白Aubergine、精氨酸蛋白甲基转移酶（PRMT5）及其辅因子、Tudor蛋白和Vasa RNA解旋酶。虽然果蝇种质装配的遗传途径已得到很好的表征，但种质蛋白的相互关系和功能还不清楚。种质还含有RNA，RNA被认为是生殖细胞特化所必需的，但其身份大多未知。果蝇生殖细胞发育和生殖细胞特化所需的大多数基因在其他动物（如小鼠）的生殖细胞中具有进化保守的作用。 我们的研究揭示了重要的种质成分的重要关系和功能。我们发现，Piwi蛋白的精氨酸甲基化，由PRMT5催化，是一种进化上保守的修饰，介导与Tudor蛋白的相互作用。我们还发现，Vasa及其小鼠同系物，含有甲基化的精氨酸。在目标1中，我们将在果蝇中功能性地表征piRNP复合物及其RNA靶标。在目标2中，我们将在小家鼠中功能性地表征piRNP复合物及其RNA靶标。研究这两种生物将突出piRNP功能和生殖细胞特化和发育的保守作用和途径，但也可能揭示物种特异性功能。 本提案中详细介绍的研究将解决piRNP和相关蛋白及其RNA靶标功能的基本方面，以及这些因子在生殖细胞特化和种系发育中的作用。

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.