Cap-Dependency in Hematopoietic Stem and Progenitor Cell Translation

造血干细胞和祖细胞翻译中的帽依赖性

• 批准号: 10228305
• 负责人: Michael Mazzola
• 金额: $ 3.96万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228305
• 关键词: 5' Untranslated Regions; Agar; Bacterial Infections; Biological Assay; Blood; Bone Marrow Transplantation; Bypass; Cell Cycle; Cell Differentiation process; Cell Line; Cell Survival; Cell physiology; Cells; Cellular Stress; Cellular Stress Response; Characteristics; Collection; Complex; Cues; Decision Making; Dependence; Eukaryotic Initiation Factor-4F; Eukaryotic Initiation Factors; Flow Cytometry; Fluorescence; Genes; Genetic Transcription; Glean; Goals; Growth; Guanosine Triphosphate; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Immune; Immunoprecipitation; Infection; Inflammation; Initiator Codon; Internal Ribosome Entry Site; Knock-out; Longevity; Messenger RNA; Molecular; Mus; Natural regeneration; Organism; Output; Production; Protein Biosynthesis; Proteins; Pyrimidine; Recycling; Regulation; Reporter; Research; Ribosomes; Role; Scanning; Stimulus; Stress; Structure; Transcript; Translating; Translation Initiation; Translational Regulation; Translational Repression; Translations; Transplantation; Western Blotting; base; biological adaptation to stress; cell regeneration; chemotherapy; improved; insight; interest; irradiation; mRNA capping; mRNA sequencing; preservation; prevent; progenitor; programs; protein complex; recruit; regeneration potential; regenerative therapy; response; stem cell function; stem cell survival; stem cells; stress management; stressor; targeted treatment; translatome; vector

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.

项目摘要 造血干细胞和祖细胞（HSPC）在整个生命周期中产生血液和免疫细胞。 一个有机体，必须保存，特别是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 程序和生态位因子维持HSPC，很少有研究探讨转录后机制， 这对快速应激反应至关重要。翻译调节允许细胞快速转移翻译输出 通过靶向单个或多组mRNA来响应刺激，包括应激源和生长线索， 独特或共享的翻译调控基序。已知干细胞具有特征性的低总转化率， 与分化细胞相比，蛋白质合成;然而，很少有研究进一步表征亚型 HSPCs中的翻译调控基序活性。我的目标是了解HSCP如何区别地使用 稳态和应激下的翻译调控机制。我特别感兴趣的是 翻译的限速步骤：起始。大多数mRNA需要一系列真核起始因子， 在mRNA的5'端组装以募集核糖体。末端寡嘧啶（TOP）基序是一个实例 以这种帽依赖性方式调控的翻译调控基序; TOP是富含嘧啶的序列 存在于许多生长相关基因的5'UTR中。或者，一些mRNA绕过了 在5'端组装一些或全部真核起始因子，并被分类为帽非依赖性转录物。 内部核糖体进入位点（IRES）是在帽-核糖体中调节的翻译调节基序的一个实例。 独立的方式。在哺乳动物的5'UTR中已经鉴定出IRES来调节基因的翻译 对细胞存活和分化很重要;它们独特的5' UTR二级结构允许核糖体复合物 在没有部分或全部帽机制的情况下，特别是在应激条件下，将其加载到mRNA上。在本项目中， 我将比较帽依赖性（TOP）和帽非依赖性（IRES）的翻译调控基序活性， 稳态和应激下的造血，以了解它们在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-翻译基序的载体，并比较帽依赖性翻译的速率。 和稳定状态下跨造血的帽非依赖性翻译。由于翻译规则特别 与细胞应激反应相关，我的第二个目标是评估造血系统中的翻译变化， 在应对细菌感染、化疗和骨髓移植等应激时， 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 第三个目标，我将确定帽依赖性和帽非依赖性翻译速率是否预测HSPC再生， 造血最终，这项研究将从分子水平上深入了解蛋白质合成是如何被HSCP调控的， 这可能为改善再生疗法提供方向， 骨髓移植