Mechanism of piRNA-mediated recognition and silencing of foreign genes

piRNA介导的外源基因识别和沉默机制

• 批准号: 8618669
• 负责人: Heng-Chi Lee
• 金额: $ 8.99万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8618669
• 关键词: Affinity; Algorithms; Animal Testing; Animals; Applied Genetics; Base Pairing; Binding; Biochemical; Biochemical Genetics; Biogenesis; Bioinformatics; Biological Assay; Biological Process; Caenorhabditis elegans; Complex; DNA Transposable Elements; Data; Defect; Defense Mechanisms; Development; Disease; Epigenetic Process; Exhibits; Failure; Family; Fellowship; Fertility; Functional RNA; Gene Expression; Gene Expression Profile; Gene Silencing; Genes; Genetic; Genetic Determinism; Genetic Screening; Genome; Genome Stability; Genomic Instability; Goals; Heterochromatin; In Vitro; Infertility; Invaded; Lead; Malignant Neoplasms; Maps; Measures; Mediating; Mentors; MicroRNAs; Modeling; Molecular Genetics; Names; Nematoda; Nucleic Acids; Nucleotides; Organism; Pathogenesis; Phase; Play; Protein Array; Protein Family; Proteins; RNA; RNA Interference; RNA Sequences; RNA biosynthesis; RNA-Directed RNA Polymerase; Reagent; Recruitment Activity; Regulation; Reporter; Research; Role; Scanning; Seeds; Site; Small RNA; System; Testing; Transgenes; Transgenic Animals; Virus; base; forward genetics; genome sequencing; in vitro testing; in vivo; insight; member; mutant; novel; piRNA; programs; public health relevance; skills; tool

DESCRIPTION (provided by applicant): Invasive foreign nucleic acids such as transposons and viruses pose a threat to all organisms, potentially causing genome instability and disease, including cancer and infertility. Fortunately, organisms have evolved a vast array of protein and nucleic acid-based mechanisms to defend against intruders. In animals, germline-expressed small RNAs known as piRNAs interact with PIWI-clade Argonaute proteins, which together promote genome stability and fertility, in part by silencing transposable elements. Yet piRNAs exhibit remarkable sequence diversity and often map to unique sequences in the genome. In the nematode worm C. elegans, for example, tens of thousands of piRNAs are individually expressed from mini-genes and lack perfectly complementary targets elsewhere in the genome. Therefore the function of piRNAs has remained mysterious. We have recently shown that piRNAs can recognize their targets via imperfect base pair interactions and trigger stable and heritable epigenetic silencing, which can be maintained by RNAi and heterochromatin factors, independently of piRNAs. The goal of this proposal is to answer several fundamental questions regarding this RNA-mediated defense system, including: How do piRNAs recognize foreign nucleic acids? What are the key molecular and genetic determinants of piRNA targeting? How does piRNAs targeting trigger an epigenetic silencing? During the K99 (mentored) phase of this fellowship, I will develop biochemical assays and genetic reporters to understand how piRNAs recognize their targets. This information will aid the development of bioinformatics tools to predict and analyze in vivo piRNA targets. In addition, I will perform a genetic screen to identify factors required for piRNA- mediated targeting and silencing. During my R00 phase, I will further use a combination of biochemical, genetic and bioinformatics approaches to study the biogenesis of piRNAs and mechanism of piRNA-induced gene silencing. The studies proposed here will provide important insights into role of piRNAs in genome surveillance and epigenetic regulation and their role in disease pathogenesis. Furthermore, I will obtain reagents, tools and skills necessary to launch my independent research program.

描述（由申请人提供）：侵入性外核酸（例如转座和病毒）对所有生物构成威胁，可能导致基因组不稳定性和疾病，包括癌症和不育。幸运的是，生物已经进化出了大量的蛋白质和基于核酸的机制来防御入侵者。在动物中，生殖线表达的小RNA被称为PIRNAS与Piwi-Clade Argonaute蛋白相互作用，该蛋白共同促进基因组稳定性和生育能力，部分是通过沉默的转座元素。然而，PIRNA表现出显着的序列多样性，并且通常映射到基因组中的独特序列。例如，在线虫蠕虫秀丽隐杆线虫中，成千上万的piRNA是通过迷你生物单独表达的，并且缺乏基因组其他地方的完全互补靶标。因此，PIRNA的功能仍然神秘。我们最近表明，PIRNA可以通过不完美的基对相互作用来识别其目标，并触发稳定且可遗传的表观遗传沉默，可以由RNAi和异染色质因子维持，而与PIRNA无关。该提案的目的是回答有关此RNA介导的防御系统的几个基本问​​题，包括：PIRNA如何识别外国核酸？ PIRNA靶向的关键分子和遗传决定因素是什么？靶向的PIRNA如何触发表观遗传沉默？在此研究金的K99阶段（指导）阶段，我将开发生化分析和遗传记者，以了解Pirnas如何识别其目标。该信息将有助于开发生物信息学工具，以预测和分析体内PIRNA靶标。此外，我将执行一个遗传屏幕以识别 PIRNA介导的靶向和沉默所需的因素。在我的R00阶段，我将进一步利用生化，遗传和生物信息学方法的结合来研究PIRNA的生物发生和PIRNA诱导的基因沉默的机制。此处提出的研究将为PIRNA在基因组监测和表观遗传调节中的作用及其在疾病发病机理中的作用提供重要见解。此外，我将获得启动独立研究计划所需的试剂，工具和技能。