Deciphering Translational Control in Stem Cells

破译干细胞的翻译控制

基本信息

批准号：
10678297
负责人：
Julia Joung
金额：
$ 6.95万
依托单位：
WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10678297
关键词：
Affect Biogenesis CRISPR screen Candidate Disease Gene Cell physiology Clustered Regularly Interspaced Short Palindromic Repeats Code Data Development Disease FRAP1 gene Fibroblasts Genes Genetic Genetic Screening Guide RNA Homeostasis Immunoprecipitation Investigation Libraries Literature Maintenance Malignant Neoplasms Mass Spectrum Analysis Measures Mediating Messenger RNA Methods MicroRNAs Molecular Nucleic Acid Regulatory Sequences Open Reading Frames Pathway interactions Peptide Initiation Factors Polyribosomes Process Protein Biosynthesis Proteins Publishing RNA-Binding Proteins Regulatory Element Regulatory Pathway Reporter Reporter Genes Ribosomes Screening Result Site Testing Translating Translational Regulation Translational Repression Translations Untranslated Regions Work cell type gene discovery gene interaction genome-wide human disease human embryonic stem cell knock-down mRNA Translation pluripotency polysome profiling preservation protein expression ribosome profiling screening stem cell function stem cells translational potential vector

项目摘要

Project Summary Translational control is critical for a variety of cellular processes such as homeostasis and differentiation. Dysregulation of translation has been implicated in many human diseases, including cancer. Stem cells are characterized by low global protein synthesis rates, despite high levels of ribosome biogenesis, suggesting that stem cells actively repress translation. Indeed, precise translational control has been shown to be essential for maintaining pluripotency, as perturbations that increased translation rates induced differentiation in various types of stem cells. Although there are several examples of how translation is regulated, the molecular mechanisms that modulate translation, particularly in stem cells, are largely unknown. To achieve a comprehensive understanding of translational control, I propose to systematically investigate the regulatory pathways that mediate translation in stem cells and across cell types. I will begin by developing a genome-scale genetic screening platform that combines CRISPR screening with polysome profiling to identify genetic perturbations that alter translation. Compared to previous approaches that relied on reporter protein expression, the proposed screening approach measures presence of ribosomes on mRNA, which more directly and accurately quantifies translation rates. This screening approach is also more versatile and can be adapted for studying other modes of translation by exchanging the reporter gene in the vector. I will then apply this screening approach to systematically discover genes that regulate global translation levels in stem cells and fibroblasts and investigate associated mechanisms. Finally, I will focus on genes that are selectively translated using ribosome profiling and characterize the pathways that mediate selective mRNA translation. Together, my proposed work establishes a framework for systematic exploration of translational control networks and, in concert with its application in other contexts, will contribute to a comprehensive understanding of the processes controlling translation that are relevant in development and disease.

项目摘要翻译控制对于各种细胞过程至关重要，例如稳态和分化。翻译失调与包括癌症在内的许多人类疾病有关。干细胞是尽管核糖体生物发生高水平，但以低全球蛋白质合成速率为特征，表明干细胞积极抑制翻译。确实，精确的翻译控制已被证明是必不可少的维持多能性，作为增加翻译率的扰动，引起了各种类型的分化干细胞。尽管有几个示例关于翻译的调节，但分子机制这种调节翻译，尤其是在干细胞中，在很大程度上未知。实现全面的理解转化控制，我建议系统地研究监管途径介导干细胞和细胞类型中的翻译。我将首先开发基因组规模的遗传筛选平台将CRISPR筛选与多元组谱分析结合在一起，以识别遗传扰动那改变了翻译。与以前依赖报告蛋白表达的方法相比，提出的筛选方法测量mRNA上核糖体的存在，该核糖体更直接，准确地量化翻译率。这种筛选方法也更具通用性，可以适应其他模式通过交换向量中的记者基因来翻译。然后，我将采用这种筛选方法系统地发现调节干细胞和成纤维细胞中全局翻译水平的基因并研究相关机制。最后，我将专注于使用核糖体分析和表征介导选择性mRNA翻译的途径。我提出的工作共同建立了用于系统探索翻译控制网络的框架，并协同其应用在其他情况下，将有助于全面理解控制翻译的过程与发育和疾病有关。