THE IMPACT OF PIWI ASSOCIATED TRANSCRIPTS IN XENOPUS GERM CELL DEVELOPMENT

PIWI 相关转录对非洲爪蟾生殖细胞发育的影响

• 批准号: 9492924
• 负责人: NELSON C LAU
• 金额: $ 24.67万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-12 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9492924
• 关键词: Address; Affect; Animals; Binding; Biochemical; Biological Assay; Candidate Disease Gene; Cell Death; Child; Complex; DNA Transposable Elements; Development; Dissection; Embryo; Embryonic Development; Female; Fertility; Fertility Study; Foundations; Future; Gametogenesis; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genome; Genomic approach; Germ Cells; Goals; Gonadal structure; Health; Human; In Vitro; Individual; Knock-out; Knowledge; Light; Location; Modeling; Molecular Profiling; Mus; Mutate; Mutation; Oocytes; Ovary; Pathway interactions; Phenotype; Play; Production; Proteins; Rana; Regulation; Regulator Genes; Reproductive Health; Role; Small RNA; Structure of primordial sex cell; System; Testing; Transcript; Xenopus; Xenopus oocyte; experimental study; in vivo; insight; male; mutant; transcriptome; vertebrate genome

Project Summary/Abstract Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for animal fertility because they repress transposable elements (TE) and regulate germ cell-specific genes. Although vertebrate genomes encode hundreds of piRNA cluster loci, we know little about the impact and gene regulation roles of individual piRNA clusters. The reason is because Piwi pathway mutants disrupt all piRNAs and cause widespread germ cell death, thereby obscuring the dissection of functions for individual piRNA Cluster loci. Major knowledge gaps include: (1) Which genes besides TEs impact fertility and are being targeted by PIWI/piRNA complexes? (2) Will a single piRNA cluster mutation affect the production of piRNAs from other clusters? (3) How will the regulation of Piwi targets and vertebrate germ cell development be affected by mutating a single piRNA cluster? This exploratory project will address these questions by first generating a list of Xenopus Piwi- associated transcripts (PATs) and testing their mechanism of regulation in Xenopus oocyte extracts. We will then create an intergenic piRNA cluster locus mutant in Xenopus tropicalis, and examine which PATs are affected in this mutant so as to gain insight into the relationship between specific groups of piRNAs and target genes regulated by Piwi/piRNA complexes. The frog is an experimentally tractable vertebrate system for dissecting the role of individual piRNA clusters. These Xenopus studies will reveal how a single major intergenic piRNA cluster will affect gonadogenesis and embryonic development. The future characterizations of mutant phenotypes are considerations for a longer-term project after the initiation of this exploratory project. The focus of this exploratory project is to create a list of PATs, test for PAT regulation in oocyte extracts, and then create a valuable mutant that will enable precise examination of which PATs are mis-regulated. Given our foundation of expertise described in this proposal, we are confident in achieving the goals of this exploratory project.

项目概要/摘要 Piwi 蛋白和 Piwi 相互作用 RNA (piRNA) 对于动物生育能力至关重要，因为它们 抑制转座元件 (TE) 并调节生殖细胞特异性基因。尽管脊椎动物基因组 编码数百个 piRNA 簇位点，我们对其影响和基因调控作用知之甚少 单个 piRNA 簇。原因是 Piwi 通路突变体破坏了所有 piRNA 并导致 广泛的生殖细胞死亡，从而模糊了单个 piRNA 簇功能的剖析 基因座。主要的知识差距包括： (1) 除了 TE 之外，还有哪些基因影响生育能力并成为目标 通过 PIWI/piRNA 复合物？ (2)单个piRNA簇突变是否会影响piRNA的产生 其他集群？ （3）Piwi靶点和脊椎动物生殖细胞发育的调控将如何进行 受单个 piRNA 簇突变的影响吗？ 这个探索性项目将通过首先生成 Xenopus Piwi 列表来解决这些问题 相关转录本（PAT）并测试其在非洲爪蟾卵母细胞提取物中的调节机制。我们 然后将在热带非洲爪蟾中创建基因间 piRNA 簇位点突变体，并检查哪些 PAT 在此突变体中受到影响，以便深入了解特定 piRNA 组之间的关系 以及由 Piwi/piRNA 复合物调控的靶基因。青蛙是一种实验上易于驯服的脊椎动物 用于剖析单个 piRNA 簇的作用的系统。这些爪蟾研究将揭示如何 单个主要基因间piRNA簇将影响性腺发生和胚胎发育。未来 突变表型的表征是启动后长期项目的考虑因素 这个探索性项目。这个探索性项目的重点是创建 PAT 列表，测试 PAT 卵母细胞提取物中的调节，然后创建有价值的突变体，从而能够精确检查 哪些 PAT 受到了不当监管。鉴于本提案中描述的我们的专业知识基础，我们 对实现这一探索性项目的目标充满信心。