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

项目总结/摘要 核糖体生物发生在核仁中，核仁蛋白质的破坏导致 核糖体病如Treacher柯林斯综合征，并导致一系列增殖性和退行性 疾病核仁包含三个嵌套的亚区室，它们彼此相分离， 在成分、密度和功能上。最内部的纤维中心（FC）被致密的纤维包围， 组件（DFC）。核糖体RNA（rRNA）的转录发生在FC和DFC之间的界面， rRNA的成熟发生在DFC中。在最外层，颗粒组分（GC）、rRNA 掺入核糖体蛋白质以产生核糖体亚基。相分离的平衡性质意味着 在没有能量输入的情况下，相关因素集中在特定的阶段。这与 核仁的嵌套结构，建立了不同层之间的化学控制的通量。 最近的研究描述了一个建立核糖体蛋白内流的分子传递模型 变成颗粒状。然而，核糖体组装需要向外流动的rRNA，这是如何 建立，这是否在热力学控制下，以及建立这种通量的关键分子仍然存在 完全未知我的目标是揭示rRNA进入和通过细胞的机制。 核仁的颗粒状成分。 在我的初步工作中，我确定了建立rRNA通量的关键因素，并开始 在体外重建这个过程。我使用生物信息学分析和体内定位研究来确定 蛋白质核仁素作为建立rRNA流的关键蛋白。我的重组显示核仁素介导rRNA 流出DFC并进入GC。最近的文献研究和我的初步研究表明， 核仁素帮助rRNA通过GC。我的初步数据引出了一个假设， 核糖体的生物发生依赖于rRNA进入和通过颗粒组分的热力学通量 (GC)这主要是由核仁素介导的。这个假设将推动我的具体目标：1 - 确定核仁素（NCL）和rRNA复合物的生物物理特征需要建立 rRNA从致密纤维组分（DFC）流入颗粒组分（GC）， 核仁素、核磷蛋白、rRNA和rProtein的一系列相关相平衡，以确定它们是否 有助于rRNA通过颗粒组分的流动