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 核糖体的生物发生发生在核仁中，核仁蛋白的破坏导致 核糖体病变，如Treacher Collins综合征，导致一系列增生性和退行性疾病 疾病。核仁包含三个嵌套的亚室，它们彼此相分离，并且不同 在组成、密度和功能上。最内侧的纤维中心(FCS)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之间的界面上， RRNA在DFC中成熟。在最外层的阶段，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 相关因素在没有能量投入的情况下集中在特定的阶段。这一点再加上 核仁的嵌套结构，建立了热力学控制的跨不同层的通量。 最近的研究描述了一个建立核糖体蛋白内流的分子交接模型 进入颗粒状成分。然而，核糖体组装需要rRNA的向外流动，以及这是如何实现的 建立，无论这是在热力学控制下，以及建立这种流动的关键分子角色仍然存在 完全未知。我的目标是揭示建立rRNA流入和通过 核仁的颗粒状成分。 在我的前期工作中，我确定了建立rRNA通量的关键因素，并开始 在体外重建这一过程。我使用生物信息学分析和体内定位研究来确定 蛋白质核仁素是建立rRNA通量的关键蛋白质。我的重组表明核仁介导rrna。 流量从DFC流出，进入GC。最近的文献研究和我的初步研究表明 核仁素有助于建立通过GC的rRNA流量。我的初步数据得出了这样的假设 核糖体的生物发生依赖于rRNA进入和通过颗粒成分的热力学通量 (GC)，这主要是由核仁介导的。这个假设将驱动我的具体目标：1- 确定需要建立核仁素(NCL)和rRNA复合体的哪些生物物理特性 RRNA从致密纤维成分(DFC)流向颗粒成分(GC)和2-措施 核仁素、核磷蛋白、rRNA和rProtein的一系列相平衡，以确定它们是否 有助于通过颗粒成分的rRNA流量