piRNA biogenesis ruled by PIWI-TUDOR interaction

PIWI-TUDOR 相互作用控制 piRNA 生物发生

• 批准号: 9281766
• 负责人: Yohei Kirino
• 金额: $ 28.55万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9281766
• 关键词: Animals; Arginine; Binding; Biochemical; Biogenesis; Bombyx; Bombyx mori; Cell Culture System; Cell Density; Cell Line; Cells; Complex; DNA Transposable Elements; Development; Disease; Drosophila genus; Enzymes; Genes; Genome; Goals; Human; Lead; Malignant Neoplasms; Mediating; Methylation; Mitochondria; Modification; Molecular; Monitor; Mus; Nucleotides; Ovary; Pathway interactions; Phosphorus; Plasmids; Play; Precursor RNA; Process; Proteins; RNA; RNA 3' End Processing; RNA Interference; RNA Precursors; Reporting; Reproduction; Research; Role; Sequence Analysis; System; Tertiary Protein Structure; Testis; Time; Transfection; Xenopus; cancer type; dimethylarginine; experimental study; genetic element; genome integrity; novel; overexpression; public health relevance; reproductive system disorder; screening; transcriptome sequencing; tumor

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. .

描述（申请人提供）：在整个真核谱系中，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）为中心的小调节RNA途径沉默转座子和其他自私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，对于种系发育至关重要。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 复合物如何沉默转座子。 我们的研究目标是阐明piRNA的生物发生和功能，并揭示种系中PIWI/piRNA途径介导的基因组防御系统的分子机制。由于最近的报告显示人类 PIWI 在各种肿瘤中表达，我们的研究将影响了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，我们之前发现 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 的发现导致我们的假设：PIWI sDMA 的重要功能是被含有 TUDOR 结构域的蛋白质识别，并且 PIWI-TUDOR 相互作用在 piRNA 生物发生和功能中发挥重要作用。 为了阐明 PIWI sDMA 在 piRNA 途径中的功能，我们将在 BmN4（一种家蚕卵巢来源的培养细胞系）中表达包含或缺乏 sDMA 的 PIWI 蛋白，该细胞系由于 PIWI/piRNA 途径的内源表达以及使用质粒转染和 RNAi 的便利性而成为 piRNA 研究的优秀细胞系统。 PIWI 蛋白之间的定位、相互作用蛋白以及相关 piRNA 的数量和质量的比较将阐明 PIWI sDMA 及其相互作用的含有 TUDOR 结构域的蛋白在 piRNA 生物发生和功能中的作用。此外，通过系统筛选包含 Bombyx TUDOR 结构域的蛋白质，我们最近确定 BmPAPI 是负责 piRNA 生物发生的因子。我们将对 BmPAPI 蛋白进行生化、结构和功能表征，以研究 BmPAPI 涉及的 piRNA 生物发生的分子基础。此外，我们最近发现细胞与细胞的接触全局激活了 BmN4 细胞系统中的 piRNA 生物发生。我们建议利用该激活系统来观察 piRNA 簇如何转录为前体 RNA 以及前体如何加工成成熟的 piRNA。 。