Structural transitions and RNA-mediated mechanisms of LSD1

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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.