Structural transitions and RNA-mediated mechanisms of LSD1

项目摘要/摘要 这个项目的中心假设是，RNA结构和RNA-染色质相关蛋白 相互作用在维持基因组稳定性方面起着至关重要的作用。已知非编码RNA(NcRNAs) 与染色质的蛋白质成分相互作用，从而改变高阶遗传结构和基因 表情。这些RNA-蛋白质相互作用的一个子集与衰老、癌症转移和神经元有关 发展。随着基因组学方法开始确定RNA-蛋白质之间的联系及其与癌症的联系， 关于基于表观遗传的RNA结合蛋白，我们的知识中仍然存在关键的空白。RNA可以模仿 DNA，在细胞压力下形成短暂的DNA/RNA杂交物，并作为支架招募蛋白质复合体 染色质。此外，这些RNA可以与结构化和无序的蛋白质区域相互作用形成 可以进行液-液相分离的多价网络，或生物分子凝聚体。因此， 检查溶液中的RNA-蛋白质组件以及类似于致密细胞的凝聚、簇状状态 环境可以解决基本问题：1)顺序要求或结构是什么 驱动多价RNA-蛋白质相互作用形成的特征？2)RNA-蛋白质是如何 构象变化影响染色质相关蛋白的功能和活性？在这项提案中，我们 试图了解RNA是如何与赖氨酸特异性脱甲基酶-1(LSD1)相互作用的。LSD1是一个 必需的甲基化调节因子，由于其广泛的相互作用，在许多癌症中具有致癌特性 在细胞分化和DNA损伤反应通路中的网络和与RNA的关联。我们将专注于 关于含有端粒重复序列的RNA(Terra)，它与LSD1相互作用，以响应DNA损伤 染色体末端。这些RNA-LSD1相互作用参与了异染色质的调节 端粒。The co-I(Zhang)发现，端粒聚集发生在无端粒酶的癌细胞中 这一过程是通过RNA介导的相分离来驱动的。我们将使用一体化的结构和 细胞生物学方法展示特定的核酸结构如何与LSD1结合以及如何更大 核酸可能在端粒维持中起到功能上的协同调节作用。我们假设更高的阶数 RNA拓扑结构是染色质相关蛋白的重要介体。这样做的具体目的是 因此，建议1)定义内在无序的结构机制和自动调节作用 LSD1区域，2)以确定端粒中涉及的LSD1-RNA组件的作用机制 维护，以及3)确定更高阶的架构和构象重排 处于溶液和生物分子凝聚状态的Terra。这个项目的长期目标是了解 RNA结构和构象动力学如何影响LSD1介导的调控网络。结果来自 这些研究将为基于RNA的染色质生物学提供新的机制洞察力，并作为一个开始 以特定上下文的方式针对LSD1和RNA的治疗学的发展中的一个点。

Project Summary/Abstract This project is centered on the hypothesis that RNA structure and RNA-chromatin associated protein interactions play crucial roles in maintaining genome stability. It is known that non-coding RNAs (ncRNAs) interact with protein components of chromatin, thus altering the higher order genetic architecture and gene expression. A subset of these RNA-protein interactions are linked to ageing, cancer metastasis, and neuronal development. As genomic approaches begin to identify RNA-protein associations and their links to cancer, there remain key gaps in our knowledge regarding epigenetic-based RNA binding proteins. RNA can mimic DNA, form transient DNA/RNA hybrids under cell stress, and function as scaffolds to recruit protein complexes to chromatin. Further, these RNAs can interact with structured and disordered protein regions to form multivalent networks that can undergo liquid-liquid phase separation, or biomolecular condensates. Thus, examining RNA-protein assemblies in solution and in a condensed, clustered state akin to a compact cell environment can address fundamental questions: 1) What are the sequence requirements or structural features that drive formation of multivalent RNA-protein interactions? And 2) How do RNA-protein conformational changes impact the function and activity of chromatin associated proteins? In this proposal, we seek to understand how RNA interacts with the lysine specific demethylase-1 (LSD1) enzyme. LSD1 is an essential methylation regulator and has oncogenic properties in many cancers due to its vast interaction network and association with RNAs in cell differentiation and DNA damage response pathways. We will focus on the telomeric repeat containing RNA (TERRA) that interacts with LSD1 in response to DNA damage at the ends of chromosomes. These RNA-LSD1 interactions are involved in the regulation of heterochromatin at telomeres. The co-I (Zhang) has discovered that telomere clustering occurs in telomerase-free cancer cells and that this process is driven through RNA-mediated phase separation. We will use integrative structural and cell biology approaches to demonstrate how specific nuclei acid structures engage with LSD1 and how larger nucleic acids may serve as functional coregulators in telomere maintenance. We hypothesize that higher-order RNA topologies function as crucial mediators of chromatin-associated proteins. The specific aims of this proposal are thus 1) to define the structural mechanism and auto-regulatory role of an intrinsically disordered region of LSD1, 2) to identify the mechanism of action for LSD1-RNA assemblies involved in telomere maintenance, and 3) to determine the architecture and conformational rearrangements of a higher-order TERRA in solution and in a biomolecular condensate state. The long-term goal of this project is to understand how RNA structure and conformational dynamics influence LSD1-mediated regulatory networks. Results from these studies will provide new mechanistic insight into RNA-based chromatin biology and serve as a starting point in the development of therapeutics that target LSD1 and RNA in a context-specific manner.