piRNA biogenesis ruled by PIWI-TUDOR interaction

描述（由申请人提供）：在整个真核谱系中，以PIWI （P- element-Induced WImpy testis）蛋白为中心的小调控RNA途径及其相关的24~30个核苷酸（nt） PIWI相互作用RNA （piRNAs）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主要在种系中表达，对种系发育至关重要。然而，与其他小调控RNA途径相比，我们缺乏对这种基因组防御系统的机制理解。piRNA的生物发生，前体rna是如何产生、加工和成熟的，在很大程度上是未知的。同样不清楚的是PIWI/piRNA复合物是如何沉默转座子的。我们的研究目标是阐明piRNA的生物发生机制和功能，揭示PIWI/piRNA通路介导的种系基因组防御系统的分子机制。由于最近的报道显示了人类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疾病的生物医学目标。为了实现这一目标，我们之前已经发现PIWI蛋白含有进化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，我们对PIWI sdma的发现导致我们假设PIWI sdma的重要功能是被含有tudor结构域的蛋白质识别，PIWI- tudor相互作用在piRNA的生物发生和功能中发挥重要作用。为了阐明PIWI sDMAs在piRNA通路中的功能，我们将在BmN4（家蚕卵巢来源的培养细胞系）中表达含有或缺乏sDMAs的PIWI蛋白，BmN4由于其内源性表达PIWI/piRNA通路，并且便于使用质粒转染和RNAi，是研究piRNA的优秀细胞系统。比较PIWI蛋白之间的定位、相互作用蛋白以及相关piRNA的数量和质量，将阐明PIWI sdma及其相互作用的含tudor结构域蛋白在piRNA生物发生和功能中的作用。此外，通过系统筛选含有家蚕tudor结构域的蛋白，我们最近确定了BmPAPI是piRNA生物发生的因素。我们将对BmPAPI蛋白进行生化、结构和功能表征，以探讨BmPAPI参与piRNA生物发生的分子基础。此外，我们最近发现，在BmN4细胞系统中，细胞间的整体接触激活了piRNA的生物发生。我们建议利用这个激活系统来观察piRNA簇如何转录成前体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. .