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

项目摘要 核糖体的生产依赖于一种名为核仁的核细胞器。在这个结构中，核糖体DNA (RDNA)被转录成与核糖体蛋白相关的RNA转录本。核仁结构是 在包括许多癌症在内的许多人类疾病中发生改变，促使多项研究寻找 核仁形态的调节剂。虽然这些研究依赖于核仁蛋白或核糖体的分析 为了识别分子调控因子，很少有研究定义细胞周期特异的机制 调节核仁结构。此外，还没有研究检查rdna空间组织的影响。 核仁形态，尽管rdna基因座是已知的核仁组织者区域。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 这一建议的目的是(I)确定rDNA空间组织的分子调控因子和(Ii)定义 驱动细胞周期调节核仁形态的过程。中心假设是空间上的 RDNA的组织部分地受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受细胞周期调节，推动动态重组 间期核仁形态。这项研究基本原理是确定保守的 裂解酵母中rDNA和核仁组织的分子调控将为未来提供模板 对高等生物体的研究。这一中心假设将通过两个具体的目标来检验：1)识别分子 裂解酵母中rDNA空间组织的调节器；以及2)定义驱动细胞周期调节的过程 核仁形态。对于目标1，一种分析活细胞中rdna空间组织的新工具已经被 在分裂酵母中培育出来的。该工具将用于通过荧光来量化rDNA空间组织 染色体组织和DNA拓扑因素改变的候选突变体的显微镜观察。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 检测细胞周期调节的核糖体生物发生因子在间期核仁中作用的方法 形态学。这些研究检查了裂解酵母中的rDNA和核仁形态，这是一个值得注意的模型系统 因为它适用于高等生物和易于进行基因操作。要了解两国关系 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。这项研究应用了创新的成像工具，具有先进的细胞和 广泛鉴定rDNA和核仁基本分子调控因子的分子生物学技术 形态学，为人类细胞的未来研究提供了一个框架。

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.