piRNA biogenesis ruled by PIWI-TUDOR interaction

PIWI-TUDOR 相互作用控制 piRNA 生物发生

• 批准号: 9068967
• 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 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9068967
• 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 Interference; RNA Precursors; Reporting; Reproduction; Research; Role; Sequence Analysis; System; Tertiary Protein Structure; Testis; Time; Transfection; Xenopus; base; cancer type; dimethylarginine; genetic element; genome integrity; novel; overexpression; piRNA; public health relevance; reproductive system disorder; research study; 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（由申请人提供）：在整个真核生物谱系中，小型调节性RNA通路以Piwi（P-元素诱导的Wimpy testis）蛋白质及其关联24〜30核苷酸（NT）PIWI相互作用RNAS（PIRNAS）的依恋rnas（PIRNAS）保持沉默传播元素和其他自私元素，以维持基因的基因组合。 PIWI蛋白和PIRNA主要在种系中表达，对于种系发育至关重要。但是，与其他小的调节RNA途径相反，我们对该基因组防御系统缺乏机械理解。 PIRNA生物发生如何产生，处理和成熟的前体RNA在很大程度上未知。同样不清楚的是piwi/pirna络合物如何沉默的转座子。 我们的研究目的是阐明PIRNA的生物发生和功能，并发现种系中PIWI/PIRNA途径介导的基因组防御系统的分子机制。由于最近的报告表明了人类PIWI在各种肿瘤中的表达，我们的研究将影响了解与生殖疾病和癌症有关的疾病的生物医学目标。为了实现目标，我们以前已经发现PIWI蛋白包含进化保存的对称二甲基金素（SDMA）。由于已知蛋白质的都铎式结构域特异性结合与SDMA，因此我们发现PIWI SDMAS导致我们的假设，即PIWI SDMA的重要功能应通过含有Tudor-Dobain的含有蛋白质的蛋白质，Piwi-Tudor相互作用在Pirna Biogenogen和Function中具有重要的疾病。 为了阐明PIWI SDMA在PIRNA途径中的功能，我们将表达PIWI蛋白在BMN4（Bombyx Mori卵巢衍生的培养细胞系）中含有或缺乏SDMA的功能，这是Pirna研究的极好细胞系统，原因是Piwi/piwi/piwi/piwi/pirna pathway and plass and plass and plass and and and and and and and and and and and and and andna。比较PIWI蛋白质之间的定位，相互作用蛋白质以及相关PIRNA的数量和质量将阐明Piwi SDMA及其相互作用的Tudor-domain在PIRNA生物发生和功能中的相互作用的Tudor-domain的作用。此外，通过对包含蛋白质的Bombyx Tudor域的系统筛选，我们最近将BMPAPI确定为负责PIRNA生物发生的因素。我们将对BMPAPI蛋白进行生化，结构和功能特征，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 - 细胞接触全球激活BMN4细胞系统中的PIRNA生物发生。我们建议利用该激活系统观察如何将PIRNA簇转录为前体RNA以及如何将前体处理成成熟的PIRNA。 。