THE IMPACT OF PIWI ASSOCIATED TRANSCRIPTS IN XENOPUS GERM CELL DEVELOPMENT

项目摘要/摘要 Piwi蛋白和Piwi相互作用RNA(PiRNAs)对动物的繁殖力是必不可少的，因为它们 抑制转座元件(TE)，调节生殖细胞特异性基因。尽管脊椎动物的基因组 编码数百个piRNA簇基因座，我们对其影响和基因调控作用知之甚少 单个的piRNA簇。原因是Piwi途径突变破坏了所有的piRNA，并导致 广泛的生殖细胞死亡，从而掩盖了对单个piRNA簇功能的剖析 精神错乱。主要的知识差距包括：(1)除TES外，哪些基因影响生育能力，并被作为靶点 通过piwi/piRNA复合体？(2)单个piRNA簇突变是否会影响来自 其他集群？(3)Piwi靶标和脊椎动物生殖细胞发育的调控将如何 受单个piRNA簇突变的影响？ 这个探索性项目将通过首先生成非洲爪哇-皮威的名单来解决这些问题- 相关转录本(PATs)及其在非洲爪哇卵母细胞提取物中的调控机制。我们 然后，我将在热带非洲爪哇中创建一个基因间piRNA簇位点突变，并检查哪些PATS 在这个突变体中受到影响，以便深入了解特定的piRNA组之间的关系 以及受Piwi/piRNA复合体调控的靶基因。青蛙是一种实验上容易驯服的脊椎动物 用于剖析单个piRNA簇的作用的系统。这些非洲爪哇的研究将揭示一个 单个主要的基因间piRNA簇会影响性腺发生和胚胎发育。未来 突变表型的特征是在启动后的长期项目中的考虑因素 这个探索性的项目。此探索性项目的重点是创建一个PAT列表，测试PAT 调控卵母细胞提取液，然后创造一个有价值的突变体，使精确检测 哪些拍子被错误地监管。鉴于我们在本提案中所描述的专业知识基础，我们 有信心实现这一探索性项目的目标。

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.