Cap-Dependency in Hematopoietic Stem and Progenitor Cell Translation

项目摘要 造血干细胞和祖细胞(HSPC)在人的整个生命周期中产生血液和免疫细胞 一种有机体，必须保存，尤其是为了在感染等压力下生存和发挥作用， 炎症和骨髓移植。虽然广泛的研究已经阐明了转录 程序和利基因素维持HSPC，很少有研究探索转录后机制， 这对于快速的应激反应至关重要。翻译调节允许细胞快速转换翻译输出 作为对刺激的反应，包括应激源和生长线索，通过靶向单个或组基于 独特或共享的翻译规范主题。众所周知，干细胞的全细胞率非常低。 蛋白质合成与分化细胞的比较；然而，很少有研究进一步表征亚型 翻译调控基序在HSPC中的活性。我的目标是了解HSCP如何与众不同地使用 稳态和应激状态下的翻译调控机制。我特别感兴趣的是对 翻译的限速步骤：启动。大多数mRNAs需要一组真核启动因子来 在信使核糖核酸的5‘端聚集，以招募核糖体。末端寡嘧啶(顶部)基序就是一个例子 以这种帽子依赖的方式调节的翻译调控基序；顶端是富含嘧啶的序列 存在于许多生长相关基因的5‘端非编码区。或者，一些mRNA可以绕过这种需要 在5‘端组装一些或全部真核启动因子，被归类为帽非依赖性转录本。 内部核糖体进入位点(IRESS)是翻译调控基序的一个例子，在帽- 以独立的方式。已在哺乳动物的5‘UTRs中发现了调节基因翻译的IRES 对细胞存活和分化很重要；其独特的5‘UTR二级结构使核糖体复合体 在没有部分或全部帽子机械的情况下加载到mRNA上，尤指在压力条件下。在这个项目中， 我将比较大写依赖(Top)和大写非依赖(IRES)的翻译调控基序活动 稳定状态和应激状态下的造血功能，了解其在HSPC功能和存活中的作用。在我的第一个目标， 我将创建比较几个IRES和TOP翻译基序的矢量，并比较依赖大小写的比率 稳定状态下跨造血系统的帽子非依赖性翻译。由于翻译法规特别是 与细胞应激反应相关，我的第二个目标是评估整个造血过程中的翻译变化 通过评估对细菌感染、化疗和骨髓移植等应激反应 报告活性和进行核糖体免疫沉淀以确定翻译组的变化。在我的 第三个目标，我将确定大写依赖和大写无关的转换率是否可以预测HSPC的再生 造血术。最终，这项研究将收集关于蛋白质合成如何受到HSCP调控的分子洞察力， 这可能为改进骨骼等应激性体外操作的再生治疗提供方向 骨髓移植。

Project Abstract Hematopoietic stem and progenitor cells (HSPCs) produce blood and immune cells throughout the lifespan of an organism and must be preserved, especially to survive and function under stresses like infection, inflammation, and bone marrow transplantation. While extensive research has elucidated transcriptional programs and niche factors that maintain HSPCs, few studies have explored post-transcriptional mechanisms, which are critical for rapid stress responses. Translation regulation allows cells to quickly shift translational output in response to stimuli, including stressors and growth cues, by targeting single or groups of mRNAs based on unique or shared translation regulatory motifs. Stem cells are known to have characteristically low rates of total protein synthesis in comparison to differentiated cells; however, few studies have further characterized subtypes of translational regulatory motif activity in HSPCs. My goal is to understand how HSCPs differentially employ mechanisms of translational regulation in steady state and stress. I am particularly interested in regulation of the rate-limiting step of translation: initiation. Most mRNAs require a collection of eukaryotic initiation factors to assemble at the 5’ cap of an mRNA to recruit ribosomes. Terminal oligopyrimidine (TOP) motifs are one example of translation regulatory motifs regulated in this cap-dependent manner; TOPs are pyrimidine-rich sequences that exist in the 5’ UTRs of many growth-associated genes. Alternatively, some mRNAs bypass the need to assemble some or all eukaryotic initiation factors at the 5’ cap and are classified as cap-independent transcripts. Internal ribosome entry sites (IRESs) are one example of a translation regulatory motif regulated in a cap- independent manner. IRESs have been identified in mammalian 5’ UTRs to regulate the translation of genes important for cell survival and differentiation; their unique 5’ UTR secondary structures allow ribosome complexes to load onto mRNAs without some or all of the cap-machinery, especially under stress conditions. In this project, I will compare cap-dependent (TOP) and cap-independent (IRES) translation regulatory motif activity across hematopoiesis in steady state and stress to understand their role in HSPC function and survival. In my first aim, I will create vectors that compare several IRES- and TOP-translation motifs and compare rates of cap-dependent and cap-independent translation across hematopoiesis in steady state. Since translation regulation is particularly relevant to the cellular stress response, my second aim will assess translational changes across hematopoiesis in response to stresses like bacterial infection, chemotherapy, and bone marrow transplantation by assessing reporter activity and performing ribosome-immunoprecipitations to determine changes in the translatome. In my third aim, I will determine if cap-dependent and cap-independent translation rates predict HSPC regeneration of hematopoiesis. Ultimately, this study will glean molecular insight into how protein synthesis is regulated HSCPs, which may offer directions for improving regenerative therapies with stressful ex vivo manipulations like bone marrow transplants.