Deciphering Translational Control in Stem Cells

项目摘要 翻译控制对于多种细胞过程如稳态和分化是至关重要的。 翻译失调与许多人类疾病有关，包括癌症。干细胞是 尽管核糖体生物合成水平高，但其特征是整体蛋白质合成率低，这表明， 干细胞主动抑制翻译。事实上，精确的翻译控制已被证明是必不可少的， 维持多能性，因为增加翻译速率的扰动诱导了各种类型的分化， 干细胞虽然有几个例子说明翻译是如何调节的，但分子机制 特别是在干细胞中，调节翻译的基因在很大程度上是未知的。实现全面 理解的翻译控制，我建议系统地研究调控途径， 介导干细胞和跨细胞类型的翻译。我将开始开发一个基因组规模的遗传学 筛选平台，将CRISPR筛选与多核糖体分析相结合，以识别遗传扰动 改变翻译。与以前依赖于报告蛋白表达的方法相比， 筛选方法测量mRNA上核糖体的存在，其更直接和准确地定量 翻译率。这种筛选方法也更通用，可适用于研究其他模式 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 系统地发现在干细胞和成纤维细胞中调节全局翻译水平的基因，并研究 相关机制。最后，我将重点介绍使用核糖体分析选择性翻译的基因， 表征介导选择性mRNA翻译的途径。总之，我的工作计划 翻译控制网络的系统探索框架，并与其应用相一致 在其他情况下，将有助于全面了解控制翻译的过程， 与发育和疾病有关。

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.