Mechanisms of Genomic Stability by Mammalian Argonaute Proteins

项目概要/摘要 基因组的不稳定性增加了突变、染色体重排和非整倍体的发生率， 许多与年龄有关的人类疾病，包括癌症。复制我们的基因组的挑战， 表观基因组与每个细胞分裂需要分子途径，确保稳定的基因组的繁殖 子细胞。哺乳动物非编码RNA（ncRNA）在基因组维持中的作用 对稳定性的理解不完全。我们认为，填补这一空白是必要的，以扩大我们的知识， 人类发展和体内平衡，并确定新的危险因素，有助于人类疾病。在 在裂殖酵母中，RNA干扰（RNAi）途径在DNA合成期间作用于以下重复序列： 通过局部产生的ncRNA来调节着丝粒周围的细胞，以建立基因组稳定性所需的异染色质。它 目前还不清楚在哺乳动物中是否存在类似的细胞周期特异性RNAi基因组稳定性机制。在 在初步研究中，我们使用小鼠干细胞系统来绘制RNAi的高置信度相互作用， Argonaute（Ago）效应蛋白与ncRNA。我们确定Ago直接结合于着丝粒周围 ncRNA。此外，我们发现，在DNA合成过程中， Ago缺陷的细胞，并观察到由完全Ago耗尽引起的基因组不稳定性的多个特征。在 与此同时，先前在小鼠模型中的研究已经确定，着丝粒周围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调节建立的近着丝粒ncRNA表达对于 维持哺乳动物细胞中的基因组稳定性，类似于裂殖酵母。在这个RO 1研究项目中， 我们研究了RNAi在防止由积累引起的基因组不稳定性方面的潜在作用， 着丝粒周围ncRNA和/或局部异染色质结构的破坏。我们建议定义细胞周期- 特异性Ago活性，以阐明着丝粒周围调节的分子触发因素，并确定如何 近着丝粒ncRNA有助于基因组稳定性。在目标1中，我们将确定如何以及何时开始 在近着丝粒处起作用以建立异染色质。在目标2中，我们确定直接 Ago募集到染色质的分子结果。最后，在目标3中，我们将阐明ncRNA如何 在细胞周期进程中控制着丝粒周围的Ago活性。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 着丝粒周围异染色质和基因组稳定性，并将在我们对RNA的理解中开辟新的天地- 介导的基因调控我们预测这些研究将推动并产生未来战略的框架， 在疾病环境中调节着丝粒周围ncRNA。

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.