Uncovering mechanisms of rRNA flux through the nucleolus

揭示 rRNA 通过核仁的流动机制

• 批准号: 10538003
• 负责人: Matthew R King
• 金额: $ 6.76万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10538003
• 关键词: Architecture; Binding; Biochemical; Biogenesis; Bioinformatics; Biophysics; Cell Nucleolus; Clinical; Complex; Computer Models; Coupled; Data; Degenerative Disorder; Disease; Electron Microscopy; Equilibrium; Genetic Transcription; Goals; Grain; In Vitro; Inherited; Investigation; Knowledge; Lead; Life; Link; Literature; Malignant Neoplasms; Mandibulofacial Dysostosis; Measures; Mediating; Membrane; Methods; Microscopy; Modeling; Molecular; Molecular Target; Muscular Dystrophies; Names; Nature; Nucleic Acids; Nucleolar Proteins; Organelles; Phase; Physiology; Play; Process; Production; Proteins; RNA; RNA Splicing; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Series; Set protein; Structure; System; Testing; Therapeutic; Thermodynamics; Work; base; biophysical properties; cell growth; density; in vivo; molecular scale; new therapeutic target; nucleolin; nucleophosmin; reconstitution; therapeutic target

PROJECT SUMMARY / ABSTRACT Ribosome biogenesis occurs in the nucleolus, and disruptions to nucleolar proteins cause ribosomopathies such as Treacher Collins syndrome and contribute to a range of proliferative and degenerative diseases. Nucleoli contain three nested sub-compartments that are phase-separated from each other and differ in composition, density, and function. The inner-most fibrillar centers (FCs) are surrounded by dense fibrillar components (DFCs). Transcription of ribosomal RNA (rRNA) occurs at the interface between the FC and DFC, and maturation of rRNA occurs in the DFC. In the outermost phase, the granular component (GC), rRNA incorporates ribosomal proteins to create ribosomal subunits. The equilibrium nature of phase separation implies that the relevant factors concentrate themselves into specific phases without energy input. This coupled with the nested architecture of the nucleolus, sets up thermodynamically controlled fluxes across the distinct layers. Recent studies have described a molecular handoff model for setting up the inward flux of ribosomal proteins into the granular component. However, ribosomal assembly requires an outward flux of rRNA, and how this is set up, whether this is under thermodynamic control, and the key molecular players that set up such a flux remain entirely unknown. My goal is to uncover the mechanisms that set up the flux of rRNA into and through the granular component of the nucleolus. In my preliminary work I determined the critical factors involved in setting up rRNA flux and began reconstituting this process in vitro. I used bioinformatics analysis and in vivo localization studies to identify the protein nucleolin as the key protein that sets up rRNA flux. My reconstitutions show that nucleolin mediates rRNA flux out of the DFC and into the GC. Recent studies in the literature and my preliminary studies show that nucleolin helps set up the flux of rRNA through the GC. My preliminary data leads to the hypothesis that ribosome biogenesis relies on the thermodynamic flux of rRNA into and through the granular component (GC) and that this is primarily mediated by nucleolin. This hypothesis will drive my specific aims: 1 - Determine which biophysical features of nucleolin (NCL) and rRNA complexes are required to set up rRNA fluxes from the dense fibrillar component (DFC) into the granular component (GC) and 2 - measure a series of linked phase equilibria of nucleolin, nucleophosmin, rRNA, and rProtein to determine if they contribute to rRNA flux through the granular component

项目摘要 /摘要 核糖体生物发生发生在核仁中，对核酸蛋白的破坏引起 核糖体病（例如Treacher Collins综合征），并导致一系列增殖和退化 疾病。 Nucleoli包含三个嵌套的子组件，它们相互分离并不同 在组成，密度和功能中。最内部的纤维中心（FCS）被密集的纤维围绕 组件（DFCS）。核糖体RNA（rRNA）的转录发生在FC和DFC之间的界面上 rRNA的成熟发生在DFC中。在最外面的相位，颗粒成分（GC），rRNA 结合核糖体蛋白来创建核糖体亚基。相分离的平衡性质意味着 相关因素将自己集中在没有能量输入的特定阶段。这与 核仁的嵌套结构在不同层上建立了热力学控制的通量。 最近的研究描述了一种分子交接模型，用于建立核糖体蛋白质的内通量 进入颗粒成分。但是，核糖体组装需要外部rRNA，以及如何 设置，无论是在热力学控制之下，设置如此通量的关键分子玩家仍然存在 完全未知。我的目标是揭示建立rRNA磁通的机制 核仁的颗粒成分。 在我的初步工作中，我确定了设置rRNA通量涉及的关键因素并开始 在体外重新建立此过程。我使用生物信息学分析和体内定位研究来确定 蛋白质核素作为建立rRNA通量的关键蛋白质。我的重组表明核仁蛋白介导rRNA 从DFC进入GC。文献的最新研究和我的初步研究表明 核素有助于通过GC建立rRNA的通量。我的初步数据导致了以下假设 核糖体生物发生依赖于rRNA的热力学通量进入颗粒成分 （GC），这主要是由核醇蛋白介导的。这个假设将推动我的具体目标：1- 确定需要建立核仁素（NCL）和rRNA复合物的生物物理特征 来自密集的原纤维成分（DFC）的rRNA通量到颗粒成分（GC）和2-测量 一系列连接的核酸素，核素，rRNA和rprotein的相连相平衡，以确定它们是否是否 通过颗粒分量有助于rRNA通量