Deciphering Translational Control in Stem Cells

破译干细胞的翻译控制

• 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
• 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

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 翻译控制对多种细胞过程至关重要，如动态平衡和分化。 翻译的失调与包括癌症在内的许多人类疾病有关。干细胞是 具有全球蛋白质合成率低的特点，尽管核糖体生物发生水平很高，这表明 干细胞主动抑制翻译。事实上，精确的平移控制已被证明是必不可少的 保持多能性，因为增加翻译率的扰动诱导了各种类型的分化 干细胞。尽管有几个例子说明了翻译是如何被调控的，但分子机制 对翻译的调节，特别是在干细胞中，很大程度上是未知的。要实现全面的 对于翻译控制的理解，我建议系统地研究 调节干细胞和不同细胞类型之间的翻译。我将首先开发一种基因组规模的基因 结合CRISPR筛查和多聚体图谱以识别遗传扰动的筛查平台 那就是改变翻译。与以前依赖报告蛋白表达的方法相比，建议的 筛选方法测量mRNA上核糖体的存在，这更直接和准确地定量 翻译率。这种筛选方法也更具通用性，也可以适用于其他模式的研究 通过交换载体中的报告基因进行翻译。然后，我将把这种筛选方法应用于 系统地发现干细胞和成纤维细胞中调节全球翻译水平的基因，并研究 相关机制。最后，我将重点介绍使用核糖体分析和 描述介导选择性信使核糖核酸翻译的途径。总之，我提议的工作确立了 平移控制网系统探测框架及其应用 在其他情况下，将有助于全面了解控制翻译的流程 与发育和疾病有关。