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Molecular Regulation of Stem Cell Quiescence

干细胞静止的分子调控

基本信息

批准号：
10400013
负责人：
THOMAS A. RANDO
金额：
$ 4万
依托单位：
PALO ALTO VETERANS INSTIT FOR RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-01 至 2022-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10400013
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 Gene Expression Profile 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 regeneration potential repaired response response to injury sample fixation satellite cell stem cell population stem cells transcriptome transcriptome sequencing transcriptomics translatome uracil analog

项目摘要

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.

项目概要干细胞负责体内大多数组织的稳态和修复。许多人口的干细胞持续处于静止状态，直到受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关注静止肌肉干细胞（MuSC）的一些意想不到的特征，其中许多是其他静止干细胞群。这些包括积极维持细胞静止、独特的静止和激活干细胞的代谢和能量机制，以及大量存在未检测到蛋白质产物的转录本。后一个观察提出了三个主要问题：该提案的重点是：1）MuSC（或任何其他干细胞群）的转录谱是否存在于体内与通过荧光激活细胞分选（FACS）分离和纯化的细胞相似吗？ 2) 转录后机制导致蛋白质产物缺失的原因是什么转录本存在于静止细胞中吗？ 3）这些蛋白质的积累会产生什么后果静止细胞中的产物是否需要机制来防止这种积累？为了解决这些问题，该提案分为以下三个具体目标。目标1：研究静态和激活 MuSC 转录组的动态。我们将使用方法来标记新生RNA 体内（使用 4-硫尿嘧啶 (TU) 和 5-乙炔基尿苷 (EU)），然后进行标记转录物纯化和 RNA- seq 评估 MuSC 体内和离体转录动态。我们还将使用 RNA-seq 分析转录本固定 MuSC 以评估体内转录本丰度。目标 2：研究 MuSC 的翻译组和蛋白质组体内和离体。我们将分离核糖体相关转录本（使用 RiboTag 小鼠），然后对标记蛋白进行 RNA-seq 和 OP-嘌呤霉素标记，然后进行质谱分析以评估转录本与多核糖体相关并在体内翻译成静态 MuSC 中可检测的蛋白质和离体。我们还将在 MuSC 过程中体内和离体评估 MuSC 中的蛋白质翻译激活。目标 3：了解 MyoD 翻译的调控以及异常 MyoD 的后果静态 MuSC 中的蛋白质表达。根据初步数据，我们将检验以下假设：Staufen1 抑制静止细胞中 MyoD 转录物的翻译，我们将分析其功能通过抑制这些抑制机制，静态 MuSC 中 MyoD 表达的后果遗传并使用 siRNA 方法。这些研究的总体目标是提供更准确的评估静止干细胞的体内状态并更详细地了解分子维持干细胞静止并同时为细胞激活做好准备的机制差异化。