Dissection of the piRNA biogenesis pathway in germ cells

项目总结/摘要 在整个真核细胞谱系中，小的调节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）的PIWI相互作用 RNA（piRNA）沉默转座子和其他自私的遗传元件，以保持基因组的完整性。 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。 发展然而，与其他小的调控RNA途径相比，我们缺乏一种机制， 了解这个基因组防御系统。皮尔纳的生物起源如何前体RNA是 产生、加工和成熟的过程在很大程度上是未知的。同样不清楚的是PIWI/皮尔纳如何 复合沉默转座子。 我们的研究目标是阐明piRNA的生物起源和功能，并揭示piRNA的分子机制。 生殖系中PIWI/皮尔纳通路介导的基因组防御系统的机制。以来 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 了解与生殖障碍和癌症有关的疾病的生物医学目标。朝向 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结构域特异性地 结合到sDMA，我们的PIWI sDMA的发现导致我们的假设， PIWI sDMA将被含有帝舵结构域的蛋白质识别，并且PIWI-帝舵结构域将被含有蛋白质的蛋白质识别。 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，我们将表达PIWI蛋白 在BmN 4（来源于家蚕卵巢的培养细胞系）中含有或缺乏sDMA， 由于其PIWI/皮尔纳的内源性表达，因此是用于皮尔纳研究的极佳细胞系统 质粒转染和RNA干扰的方便性。本地化比较， 相互作用的蛋白质，以及PIWI蛋白质之间的相关piRNA的数量和质量将 阐明PIWI sDMA及其相互作用的含TUDOR结构域的蛋白在皮尔纳中的作用 生物起源和功能。此外，通过系统地筛选Mexix TUDOR结构域， 含有蛋白质，我们最近已经确定BmPAPI作为负责皮尔纳的因子 生物起源我们将对BmPAPI进行生化、结构和功能表征 蛋白质，以研究BmPAPI参与的皮尔纳生物发生的分子基础。另外我们 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 我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。 .

Project Summary/Abstract 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. .