piRNA biogenesis ruled by PIWI-TUDOR interaction

描述（由申请人提供）：在整个真核细胞谱系中，小调控RNA途径以PIWI（P-元件诱导的WImpy睾丸）蛋白及其相关的24~30个核苷酸（nt）PIWI相互作用RNA（piRNA）沉默转座子和其他自私的遗传元件以维持基因组完整性。PIWI蛋白和piRNA主要在生殖系中表达并且对于生殖系发育是必需的。然而，与其他小的调控RNA途径相比，我们缺乏对这种基因组防御系统的机制性理解。皮尔纳生物发生如何产生、加工和成熟前体RNA在很大程度上是未知的。同样不清楚的是PIWI/皮尔纳复合物如何沉默转座子。 我们的研究目标是阐明piRNA的生物起源和功能，并揭示生殖细胞中PIWI/皮尔纳通路介导的基因组防御系统的分子机制。由于最近的报道显示人类PIWI在各种肿瘤中的表达，我们的研究将影响理解与生殖障碍和癌症相关的疾病的生物医学目标。为了实现这一目标，我们先前已经发现PIWI蛋白含有进化上保守的对称二甲基精氨酸（sDMA）。由于已知蛋白质的TUDOR结构域与sDMA特异性结合，因此我们发现PIWI sDMA导致我们假设PIWI sDMA的重要功能是由含有TUDOR结构域的蛋白质识别，并且PIWI-TUDOR相互作用在皮尔纳生物发生和功能中起重要作用。 为了阐明PIWI sDMA在皮尔纳途径中的功能，我们将在BmN 4（家蚕卵巢衍生的培养细胞系）中表达含有或缺乏sDMA的PIWI蛋白，BmN 4由于其PIWI/皮尔纳途径的内源性表达以及使用质粒转染和RNAi的便利性而成为用于皮尔纳研究的优良细胞系统。PIWI蛋白之间相关piRNA的定位、相互作用蛋白以及数量和质量的比较将阐明PIWI sDMA及其相互作用的含TUDOR结构域的蛋白在皮尔纳生物发生和功能中的作用。此外，通过系统地筛选含有BmPAPI结构域的蛋白质，我们最近已经鉴定出BmPAPI是负责皮尔纳生物合成的因子。我们将对BmPAPI蛋白进行生化、结构和功能表征，以研究BmPAPI参与的皮尔纳生物发生的分子基础。此外，我们最近发现细胞-细胞接触在BmN 4细胞系统中全面激活皮尔纳生物合成。我们建议利用这种激活系统来观察皮尔纳簇如何转录为前体RNA以及前体如何加工成成熟的piRNA。.

DESCRIPTION (provided by applicant): Throughout the eukaryotic lineage, the small regulatory RNA pathway centered on PIWI (P- element-Induced WImpy testis) proteins and their associating 24~30 nucleotides (nt) PIWI-interacting RNAs (piRNAs) silence transposons and other selfish genetic elements to maintain genome integrity. PIWI proteins and piRNAs are expressed predominantly in the germline and essential for germline development. However, in contrast to other small regulatory RNA pathways, we lack a mechanistic understanding of this genome defensive system. The piRNA biogenesis how precursor RNAs are generated, processed and matured are largely unknown. Equally unclear is how the PIWI/piRNA complexes silence transposons. Our research goal is to clarify the biogenesis and function of piRNAs, and to uncover the molecular mechanism of the PIWI/piRNA pathway-mediated genome defensive system in the germline. Since recent reports have shown the human PIWI expression in various tumors, our research will impact biomedical goals of understanding diseases related to reproduction disorders and cancers. Toward the goal, we have previously discovered that PIWI proteins contain evolutionarily-conserved symmetrical dimethylarginines (sDMAs). Since TUDOR-domains of proteins are known to specifically bind to sDMAs, our discovery of PIWI sDMAs lead to our hypothesis that the important function of PIWI sDMAs is to be recognized by TUDOR-domain containing proteins, and the PIWI-TUDOR interactions play important roles in piRNA biogenesis and function. To elucidate the function of PIWI sDMAs in the piRNA pathway, we will express PIWI proteins containing or lacking sDMAs in BmN4 (a Bombyx mori ovary-derived cultured cell line) that is an excellent cell system for piRNA research due to its endogenous expression of the PIWI/piRNA pathway and convenience to use plasmid transfection and RNAi. Comparison of localization, interacting proteins, and quantity and quality of the associated piRNAs between the PIWI proteins will clarify the role of the PIWI sDMAs and their interacting TUDOR-domain containing proteins in piRNA biogenesis and function. Moreover, by systematical screening for the Bombyx TUDOR-domain containing proteins, we have recently identified BmPAPI as the factor responsible for piRNA biogenesis. We will perform biochemical, structural and functional characterizations on BmPAPI protein to investigate the molecular basis of BmPAPI-involved piRNA biogenesis. In addition, we recently found that cell-cell contact globally activates the piRNA biogenesis in BmN4 cell system. We propose to utilize this activation system to observe how piRNA clusters are transcribed to precursor RNAs and how the precursors are processed into mature piRNAs. .