Molecular Regulation of Stem Cell Quiescence

干细胞静止的分子调控

• 批准号: 10174500
• 负责人: THOMAS A. RANDO
• 金额: $ 5.82万
• 依托单位: PALO ALTO VETERANS INSTIT FOR RESEARCH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10174500
• 关键词: 4-thiouracil; Address; Amanitins; Attention; Cell Cycle; Cell Separation; Cells; Characteristics; Complement; Data; Environment; Exhibits; Fluorescence; Fluorescence-Activated Cell Sorting; Gene Expression; Genetic; Genetic Models; Genetic Transcription; Goals; Homeostasis; Impairment; Injury; Label; Maintenance; Mass Spectrum Analysis; Metabolic; Methods; Molecular; Mus; Muscle; Muscle satellite cell; MyoD Protein; Natural regeneration; Physiologic pulse; Polyribosomes; Population; Post-Transcriptional Regulation; Process; Production; Proliferating; Proteins; Proteome; Puromycin; RNA; RNA Synthesis Inhibitors; RNA-Binding Proteins; Regulation; Research; RiboTag; Ribosomes; Role; Skeletal Muscle; Small Interfering RNA; Stimulus; Testing; Time; Tissues; Transcript; Translating; Translational Repression; Translations; Uridine; Work; analog; base; in vivo; interest; knock-down; loss of function; next generation sequencing; novel strategies; prevent; protein degradation; protein expression; regenerative; repaired; response; response to injury; sample fixation; satellite cell; stem cell population; stem cells; transcriptome; transcriptome sequencing; transcriptomics; translatome; uracil analog

PROJECT SUMMARY Stem cells are responsible for homeostasis and repair of most of the tissues in the body. Many populations of stem cells persist in a quiescent state until stimulated to enter the cell cycle, proliferate, and differentiate into functional cells of the particular tissue. In recent years, work from our group and others has drawn attention to several unexpected characteristics of quiescent muscle stem cells (MuSCs), many of which are shared by other quiescent stem cell populations. These include the active maintenance of cellular quiescence, unique metabolic and energetic mechanism in quiescent and activating stem cells, and the presence of large numbers of transcripts for which no protein product is detected. This latter observation raises three major questions that are the focus of this proposal: 1) Is the transcriptional profile of MuSCs (or any other stem cell population) in vivo similar to that of cells that have been isolated and purified by fluorescence-activated cell sorting (FACS)? 2) What are the post-transcriptional mechanisms that are responsible for the absence of protein products when transcripts are present in the quiescent cells? 3) What are consequences of accumulation of those protein products in quiescent cells that necessitate mechanisms to prevent such an accumulation? To address these issues, this proposal is divided into three Specific Aims as follows. Aim 1: To study the dynamics of the quiescent and activating MuSC transcriptome. We will use methods to label nascent RNA in vivo (using 4-thiouracil (TU) and 5-ethynyl uridine (EU)) followed by labelled transcript purification and RNA- seq to assess MuSC transcript dynamics in vivo and ex vivo. We will also profile transcripts using RNA-seq of fixed MuSCs to assess transcript abundance in vivo. Aim 2: To study the translatome and proteome of MuSCs in vivo and ex vivo. We will isolate ribosome-associated transcripts (using the RiboTag mouse) followed by RNA-seq and OP-puromycin labelling of labelled proteins followed by mass spectrometry to assess transcripts that are associated with the polyribosome and translated into detectable proteins in quiescent MuSCs in vivo and ex vivo. We will also assess protein translation in MuSCs in vivo and ex vivo during the process of MuSC activation. Aim 3: To understand the regulation of MyoD translation and the consequences of aberrant MyoD protein expression in quiescent MuSCs. Based on Preliminary Data, we will test the hypothesis that Staufen1 suppresses the translation of the MyoD transcript in quiescent cells, and we will analyze the functional consequences of MyoD expression in quiescent MuSCs by inhibiting those suppressive mechanisms genetically and using an siRNA approach. The overall goals of these studies are to provide a more accurate assessment of the in vivo state of the quiescent stem cell and to understand in greater detail the molecular mechanisms that maintain stem cell quiescence and at the same time prime the cell for activation and differentiation.

项目摘要 干细胞负责体内稳态并修复体内大多数组织。许多人口 干细胞的持续状态一直处于静止状态，直到刺激进入细胞周期，增殖并分化为 特定组织的功能细胞。近年来，我们小组和其他人的工作引起了人们的注意 静态肌肉干细胞（MUSC）的几个意外特征，其中许多是由 其他静止的干细胞群体。这些包括主动维持细胞静止，独特 静态和激活干细胞中的代谢和能量机制，并且存在大量 未检测到蛋白质产物的转录本。后一种观察提出了三个主要问题 这是该提议的重点：1）是MUSC（或任何其他干细胞种群）的转录特征 与已通过荧光激活的细胞分选（FACS）分离和纯化的细胞相似的体内？ 2）当哪种后期机制是什么原因导致蛋白质产物的缺乏 静态细胞中存在转录本吗？ 3）这些蛋白质积累的后果是什么 静态细胞中的产物需要机制防止这种积累？ 为了解决这些问题，该提案分为三个特定目标，如下所示。目标1：研究 静止和激活MUSC转录组的动力学。我们将使用方法将新生RNA标记 Vivo（使用4-硫核（Tu）和5-乙基尿苷（EU）），然后标记为转录本纯化和RNA- SEQ在体内和体内评估MUSC转录动力学。我们还将使用RNA-seq概述成绩单 修复了MUSC，以评估体内成绩单丰度。目标2：研究MUSC的翻译组和蛋白质组 体内和前体体。我们将隔离核糖体相关的转录本（使用Ribotag鼠标），然后是 标记蛋白的RNA-Seq和Op-Puromycin标记，然后是质谱法以评估转录本 与多核糖体相关并转化为静态MUSC中可检测的蛋白 和ex vivo。我们还将在MUSC过程中评估MUSC的体内MUSC和Ex Vivo的蛋白质翻译 激活。目标3：了解Myod翻译的调节和异常Myod的后果 静态MUSC中的蛋白质表达。根据初步数据，我们将测试Staufen1的假设 抑制静态细胞中myod转录本的翻译，我们将分析功能 通过抑制这些抑制机制，静态MUSC中Myod表达的后果 遗传和使用siRNA方法。这些研究的总体目标是提供更准确的 评估静态干细胞的体内状态，并更详细地了解分子 维持干细胞静止和同时启动细胞激活和激活的机制 分化。