Dissect the piRNA regulatory mechanism during spermatogenesis

解析精子发生过程中的piRNA调控机制

• 批准号: 8925978
• 负责人: Xin Li
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-05 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8925978
• 关键词: 4-thiouracil; Architecture; Biochemical; Biogenesis; Bioinformatics; Biology; Cell-Free System; Cells; Code; Complement; Coupling; Crude Extracts; DNA Methylation; Development; Elements; Enhancers; Epigenetic Process; Event; Exons; Fertility; Fluorescent in Situ Hybridization; Foundations; Genes; Genetic Transcription; Genome; Germ Cells; Goals; Gonadal structure; Grant; High-Throughput Nucleotide Sequencing; Human; In Vitro; Infertility; Introns; Laboratories; Learning; Length; Light; Location; Male Infertility; Mammals; Meiosis; Metabolism; Molecular; Mus; Mutant Strains Mice; Nucleic Acids; Organism; Pathway interactions; Phase; Physiologic pulse; Poly(A) Tail; Process; Production; Proteins; Quantitative Microscopy; RNA; RNA Processing; RNA Splicing; Regulation; Regulatory Element; Research; Research Personnel; Role; Small RNA; Somatic Cell; Spermatocytes; Spermatogenesis; Spermiogenesis; Structure; System; Techniques; Testis; Transcript; Translations; Untranslated RNA; Uridine; deep sequencing; design; experience; histone modification; human male; male; novel strategies; piRNA; promoter; reconstitution; research study; sperm cell; transcription factor

Project Summary  PIWI proteins and their associated small RNAs (piRNAs) are essential for male fertility in mammals. My long term goal is to understand the biology of piRNAs and their function in male fertility. Unknown mechanisms determine that some RNAs are loaded into PIWI and become piRNAs, while others are not. In this grant, I propose to study the structural and mechanistic factors that determine piRNA formation. I hypothesize that both (1) the unique structure of piRNA-producing loci and (2) specific post-transcriptional processing events target a transcript for piRNA production. Yet, neither is well understood. In the K99 phase of this proposal, I will study piRNA promoters, piRNA precursors, and piRNA processing intermediates. For these studies, I will develop new strategies for deep sequencing and fluorescent in-situ hybridization.I also propose to set up a cell-free system that recapitulates piRNA production for biochemical studies. In preliminary studies, I have already defined the transcription unit of piRNA producing loci, allowing their comparison with other genes, and I have observed a fragmentation event probably responsible for 5ʹ′ end formation of piRNAs, perhaps coupling with PIWI loading. In the R00 phase of this proposal, I will identify the elements and events that are necessary and sufficient for piRNA production, and dissect the steps and locations of piRNAs metabolism in the context of spermatogenesis. The proposed study to define a piRNA will help us to understand why piRNA mutant mice are infertile, leading to a better understanding of human male infertility.

项目摘要  PIWI 蛋白及其相关小 RNA (piRNA) 对于哺乳动物的雄性生育能力至关重要。我的长 长期目标是了解 piRNA 的生物学及其在男性生育能力中的功能。未知机制 确定一些 RNA 被加载到 PIWI 中并成为 piRNA，而另一些则不是。在这笔补助金中，我 提议研究决定 piRNA 形成的结构和机制因素。我假设 (1) piRNA 产生位点的独特结构和 (2) 特定的转录后加工事件 靶向 piRNA 生产的转录本。然而，这两者都没有得到很好的理解。在这个提案的K99阶段，我将 研究 piRNA 启动子、piRNA 前体和 piRNA 加工中间体。为了这些研究，我将 开发深度测序和荧光原位杂交的新策略。我还建议建立一个 无细胞系统，可概括用于生化研究的 piRNA 生产。在初步研究中，我有 已经定义了 piRNA 产生基因座的转录单位，允许它们与其他基因进行比较，并且我 观察到可能导致 piRNA 5′ 端形成的片段化事件，可能是偶联 与 PIWI 加载。在此提案的 R00 阶段，我将确定必要的元素和事件 并足以产生 piRNA，并在以下背景下剖析 piRNA 代谢的步骤和位置 精子发生。拟议的定义 piRNA 的研究将帮助我们理解为什么 piRNA 突变小鼠 不育，从而更好地了解人类男性不育症。