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

项目摘要 核糖体的产生依赖于一种叫做核仁的核细胞器。在这个结构中，核糖体DNA 核糖体DNA（rDNA）被转录以形成与核糖体蛋白缔合的RNA转录物。核仁结构是 在包括许多癌症在内的许多人类疾病中改变，促使几项研究寻找 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， 生产，以确定分子调节剂，很少有研究已经确定细胞周期特异性机制， 调节核仁结构。此外，还没有研究探讨rDNA空间组织的影响 尽管rDNA基因座作为核仁组织者区的作用是已知的。远景目标 是为了了解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。核心假设是， rDNA的组织部分受染色体组织蛋白和核糖体生物发生的调节; 此外，这些核糖体生物合成过程是细胞周期调节的，驱动细胞的动态重组。 间期的核仁形态。这项研究的基本原理是， rDNA的分子调节剂和裂殖酵母中的核仁组织将为未来的研究提供模板。 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）鉴定分子 在裂殖酵母中rDNA空间组织的调节剂;和2）定义驱动细胞周期调节的过程 核仁形态为了实现这一目标，我们开发了一种新的分析活细胞中rDNA空间结构的工具。 是在裂变酵母中发展起来的该工具将用于通过荧光定量rDNA空间组织 显微镜在候选突变体与改变染色体组织和DNA拓扑结构的因素。这 分析将通过全基因组高通量成像屏幕来扩展，以广泛识别 rDNA空间结构目标2将应用荧光显微镜，细胞生物学和分子生物学 研究细胞周期调节核糖体生物合成因子在间期核仁中作用的方法 形态学这些研究检测了裂殖酵母中的rDNA和核仁形态， 因为它可以应用于高等生物和基因操作。为了理解这种关系 核仁形态与人类疾病之间的关系，rDNA和核仁的调控机制， 必须确定组织。这项研究采用了创新的成像工具， 分子生物学技术，以广泛地确定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。

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.