Mechanisms of Genomic Stability by Mammalian Argonaute Proteins

哺乳动物 Argonaute 蛋白的基因组稳定性机制

• 批准号: 10471922
• 负责人: JESSE R ZAMUDIO
• 金额: $ 32.76万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10471922
• 关键词: Affect; Aneuploidy; Binding; Biological; Biological Assay; CRISPR/Cas technology; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell division; Cells; Centromere; Chromatin; Chromosomal Rearrangement; DNA biosynthesis; Defect; Development; Disease; Ensure; Fission Yeast; Future; Gene Expression Regulation; Gene Silencing; Gene Silencing Pathway; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Health; Heterochromatin; Homeostasis; Human; Human Development; Knowledge; Lead; Link; Maintenance; Malignant Neoplasms; Mammalian Cell; Mammals; Maps; Mediating; Mitosis; Mitotic; Mitotic spindle; Molecular; Mus; Mutation; Northern Blotting; Pathway interactions; Protein Region; Proteins; RNA; RNA Binding; RNA Interference; RNA Interference Pathway; RNA Stability; Regulation; Reporter; Research; Research Project Grants; Risk Factors; Role; S phase; Small RNA; Structure; System; Testing; Untranslated RNA; age related; base; chromatin immunoprecipitation; daughter cell; embryonic stem cell; epigenome; experimental study; genomic signature; human disease; innovation; insight; mammalian genome; mouse development; mouse model; mutant; novel; overexpression; prevent; recruit; stem cells; tool; tumorigenesis

Project Summary/Abstract Genome instability increases the rates of mutations, chromosomal rearrangements, and aneuploidy and drives many age-related human diseases, including cancer. The challenges of replicating our genome and epigenome with each cell division requires molecular pathways that ensure the propagation of stable genomes to daughter cells. To date, the roles of mammalian noncoding RNAs (ncRNAs) in the maintenance of genome stability is incompletely understood. We propose that filling this gap is essential to expand our knowledge of human development and homeostasis and to identify novel risk factors that contribute to human disease. In fission yeast, the RNA interference (RNAi) pathway acts during DNA synthesis at the repeats of pericentromeres via locally produced ncRNAs to establish the heterochromatin needed for genome stability. It is unclear if similar cell cycle-specific RNAi mechanisms of genome stability are present in mammals. In preliminary studies, we used mouse stem cell systems to map high-confidence interactions of the RNAi effector Argonaute (Ago) proteins with ncRNAs. We determined that Ago binds directly to pericentromeric ncRNAs. Furthermore, we found that pericentromeric ncRNAs are overexpressed during DNA synthesis in Ago-deficient cells and observed multiple signatures of genomic instability caused by full Ago depletion. In parallel, previous studies in mouse models have established that overexpression of pericentromeric ncRNA suffices to cause genomic instability and tumorigenesis. Based on these intriguing findings, we hypothesize that pericentromeric ncRNA expression, established via cell cycle-specific Ago regulation, is critical for the maintenance of genomic stability in mammalian cells, analogous to fission yeast. In this RO1 research project, we examine the potential roles of RNAi in preventing genomic instability caused by accumulating pericentromeric ncRNA and/or disruption of local heterochromatin structure. We propose to define cell cycle- specific Ago activities to elucidate the molecular triggers for pericentromere regulation and to determine how pericentromeric ncRNAs contribute to genome stability. In Aim 1, we will determine how and when Ago functions at pericentromeres for the establishment of heterochromatin. In Aim 2, we determine the direct molecular consequence of Ago recruitment to chromatin. Finally, in Aim 3, we will elucidate how ncRNAs control Ago activity at pericentromeres during cell cycle progression. These innovative studies will answer the long-standing question of whether the mammalian RNAi pathway contributes to the maintenance of pericentromere heterochromatin and genome stability and will break new ground in our understanding of RNA- mediated gene regulation. We predict these studies will drive and yield a framework for future strategies to modulate pericentromeric ncRNA in disease settings.

项目摘要/摘要 基因组不稳定性增加了突变，染色体重排和非整倍性的速率和驱动器 许多与年龄有关的人类疾病，包括癌症。复制我们的基因组和 每个细胞分裂的表观基因组需要分子途径，以确保稳定基因组的传播 致子细胞。迄今为止，哺乳动物非编码RNA（NCRNA）在维持基因组中的作用 稳定性不完全理解。我们建议填补这一差距对于扩大我们对 人类发展和体内平衡，并确定有助于人类疾病的新型危险因素。在 裂变酵母，RNA干扰（RNAI）途径在DNA合成过程中起作用。 通过局部产生的NCRNA来建立基因组稳定性所需的异染色质的周围环粒。它 尚不清楚哺乳动物中是否存在类似的基因组稳定性细胞周期特异性RNAI机制。在 初步研究，我们使用小鼠干细胞系统来绘制RNAi的高信心相互作用 带有NCRNA的效应子Argonaute（AGO）蛋白。我们确定AGO直接与周围质粒结合 ncrnas。此外，我们发现在DNA合成过程中，周围粒质粒ncRNA过表达 AGO缺乏细胞并观察到由AGO耗竭引起的基因组不稳定性的多个特征。在 相似的，在小鼠模型中先前的研究已经确定了周围质粒ncRNA的过表达 足以引起基因组不稳定性和肿瘤发生。基于这些有趣的发现，我们假设 通过细胞周期特异性AGO调控确定的丁香粒ncRNA表达对 维持哺乳动物细胞中基因组稳定性，类似于裂变酵母。在这个RO1研究项目中 我们检查了RNAi在预防基因组不稳定性中的潜在作用 周围质粒ncRNA和/或局部异染色质结构的破坏。我们建议定义细胞周期 特定的AGO活性以阐明用于周围粒度调节的分子触发器，并确定如何 周围粒粒氮NCRNA有助于基因组稳定性。在AIM 1中，我们将确定如何以及何时 可在周围粒子的功能，用于建立异染色质。在AIM 2中，我们确定直接 AGO募集到染色质的分子后果。最后，在AIM 3中，我们将阐明NCRNA如何 在细胞周期进程中，对照AGO活性在周期中的周期粒细胞中。这些创新的研究将回答 长期存在的问题是哺乳动物RNAi途径是否有助于维持 上心粒粒异染色质和基因组稳定性，将在我们对RNA-的理解中打破新的基础 介导的基因调节。我们预测这些研究将推动并为将来的策略提供一个框架 调节疾病环境中的果粒颗粒NCRNA。