The identification of fundamental molecular regulators of ribosomal DNA and nucleolar organization in fission yeast

裂殖酵母核糖体 DNA 和核仁组织基本分子调节因子的鉴定

• 批准号: 10154642
• 负责人: Alexandria Jane Cockrell
• 金额: $ 3.45万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-03 至 2024-01-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10154642
• 关键词: Address; Architecture; Area; Automobile Driving; Bacterial DNA; Biogenesis; Biological Models; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Nucleolus; Cell physiology; Cells; Cellular biology; Chromosome Structures; Chromosomes; DNA; DNA analysis; DNA-Binding Proteins; Exhibits; Fission Yeast; Fluorescence Microscopy; Future; Genetic; Genetic Transcription; Genome; Goals; Human; Image; Image Analysis; Imaging Device; Interphase; Knowledge; Label; Maintenance; Malignant Neoplasms; Measures; Methods; Modeling; Molecular; Molecular Biology; Molecular Biology Techniques; Molecular and Cellular Biology; Morphology; Neurodegenerative Disorders; Nuclear; Nucleolar Organizer Region; Nucleolar Proteins; Organelles; Organism; Physiologic pulse; Process; Production; Protein Biosynthesis; Proteins; Quantitative Microscopy; RNA; RNA Processing; Research; Ribosomal DNA; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Saccharomycetales; Structure; Testing; Transcript; Variant; cell growth; deletion library; field study; genetic approach; genetic manipulation; genome-wide; high throughput analysis; human disease; imaging genetics; innovation; mutant; novel; novel strategies; nucleolar organizing region; protein complex; tool

Project Abstract Ribosome production relies on a nuclear organelle called the nucleolus. Within this structure, ribosomal DNA (rDNA) is transcribed to form RNA transcripts that associate with ribosomal proteins. Nucleolar architecture is altered in many human diseases including numerous cancers, prompting several studies to search for regulators of nucleolar morphology. While these studies rely on analysis of nucleolar proteins or ribosome production to identify molecular regulators, few studies have defined cell cycle-specific mechanisms for regulating nucleolar structure. Furthermore, no studies have examined the impact of rDNA spatial organization on nucleolar morphology despite rDNA loci’s known role as Nucleolar Organizer Regions. The long-term goal is to understand conserved regulatory mechanisms of rDNA and nucleolar organization. The overall objectives of this proposal are to (i) to identify molecular regulators of rDNA spatial organization and (ii) define the processes driving cell cycle-regulated nucleolar morphology. The central hypothesis is that the spatial organization of rDNA is regulated, in part, by chromosome organizing proteins and ribosome biogenesis; furthermore, these ribosome biogenesis processes are cell cycle-regulated, driving dynamic reorganization of nucleolar morphology during interphase. The rationale for this study is that identification of conserved molecular regulators of rDNA and nucleolar organization in fission yeast will provide a template for future research in higher organisms. This central hypothesis will be tested by two specific aims: 1) identify molecular regulators of rDNA spatial organization in fission yeast; and 2) define the processes driving cell cycle-regulated nucleolar morphology. For aim 1, a novel tool for analysis of rDNA spatial organization in live cells has been developed in fission yeast. This tool will be used to quantify rDNA spatial organization by fluorescence microscopy in candidate mutants with altered chromosome organization and DNA topology factors. This analysis will be expanded by a genome-wide high-throughput imaging screen to broadly identify regulators of rDNA spatial organization. Aim 2 will apply fluorescence microscopy, cell biology, and molecular biology approaches to examine the role of cell cycle-regulated ribosome biogenesis factors in interphase nucleolar morphology. These studies examine rDNA and nucleolar morphology in fission yeast, a model system notable for its application to higher organisms and ease of genetic manipulation. To understand the relationship between nucleolar morphology and human disease, the regulatory mechanisms behind rDNA and nucleolar organization must be identified. This study applies innovative imaging tools with advanced cellular and molecular biology techniques to broadly identify fundamental molecular regulators of rDNA and nucleolar morphology, providing a framework for future studies in human cells.

项目摘要 核糖体的产生依赖于称为核仁的核细胞器。在这个结构中，核糖体 DNA (rDNA) 被转录形成与核糖体蛋白相关的 RNA 转录物。核仁结构是 包括许多癌症在内的许多人类疾病都发生了改变，促使多项研究寻找 核仁形态的调节因子。虽然这些研究依赖于核仁蛋白或核糖体的分析 为了确定分子调节剂的生产，很少有研究定义细胞周期特异性机制 调节核仁结构。此外，没有研究检验 rDNA 空间组织的影响 尽管 rDNA 位点被称为核仁组织区域，但它对核仁形态的影响仍然存在。长期目标 目的是了解 rDNA 和核仁组织的保守调控机制。总体目标 该提案的目的是 (i) 确定 rDNA 空间组织的分子调节因子，以及 (ii) 定义 驱动细胞周期调节核仁形态的过程。中心假设是空间 rDNA 的组织部分受到染色体组织蛋白和核糖体生物发生的调节； 此外，这些核糖体生物发生过程是细胞周期调节的，驱动核糖体的动态重组 间期的核仁形态。这项研究的基本原理是鉴定保守的 裂殖酵母中 rDNA 和核仁组织的分子调节因子将为未来提供模板 高等生物的研究。这一中心假设将通过两个具体目标进行检验：1）识别分子 裂殖酵母中 rDNA 空间组织的调节因子； 2) 定义驱动细胞周期调节的过程 核仁形态。对于目标 1，一种用于分析活细胞中 rDNA 空间组织的新工具已被开发出来。 在裂殖酵母中开发。该工具将用于通过荧光量化 rDNA 空间组织 染色体组织和 DNA 拓扑因子改变的候选突变体的显微镜检查。这 分析将通过全基因组高通量成像屏幕进行扩展，以广泛识别 rDNA 空间组织。目标 2 将应用荧光显微镜、细胞生物学和分子生物学 检查细胞周期调节核糖体生物发生因子在间期核仁中的作用的方法 形态学。这些研究检查了裂殖酵母中的 rDNA 和核仁形态，这是一个值得注意的模型系统 因其在高等生物中的应用以及易于基因操作。要了解其中的关系 核仁形态与人类疾病之间、rDNA 和核仁背后的调控机制 必须确定组织。这项研究应用了具有先进细胞和技术的创新成像工具 分子生物学技术广泛鉴定 rDNA 和核仁的基本分子调节因子 形态学，为人类细胞的未来研究提供了框架。