Profiling Translational Control of Gene Expression and Nascent Proteomic Responses in Erythropoietic Progression and Ribosomopathies

分析红细胞生成进展和核糖体病中基因表达和新生蛋白质组反应的翻译控制

• 批准号: 10056119
• 负责人: CRAIG M. FORESTER
• 金额: $ 13.99万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-23 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10056119
• 关键词: Profiling; Translational; Control; Gene; Expression

SCIENTIFIC ABSTRACT Erythropoiesis is guided by a complex interplay of signaling cues triggering expression of gene networks necessary for production of mature red blood cells. Rapid response to these signals in erythroid precursors temporally orchestrate functions required for erythroid differentiation. Timely and appropriate selection of mRNAs for translation by the ribosome is essential for maintaining the balance between maintaining erythroid precursors differentiation. This concept, known as translational control governs efficiency of mRNA translation and thus plays a crucial role in regulating responses to extracellular cues such as anemia and hypoxia. Failure of the ribosomal machinery to regulate this process underlies the ribosomopathies, a set of human diseases resulting from ribosomal mutations leading to profound consequences for hematopoietic maturation. This proposal focuses on outstanding, fundamental questions in how translational control 1.) directs mRNA selection in normal erythropoiesis and 2.) is impaired in ribosomopathies in response to signaling cues. Aim 1 will explore how eIF4E, a key factor in translation initiation is repressed in a dynamic, cell-intrinsic manner to direct translation of a specific set of transcripts. Failure to repress eIF4E activity in maturing erythroblasts leads to inappropriate expression of genes necessary for maintenance of early erythroid precursors and impaired differentiation. This aim will mechanistically decipher motifs in target mRNA and decipher how eIF4E specifically recognizes these transcripts for ribosomal recruitment. Dependence of tight regulation of eIF4E on erythropoiesis will be tested in vivo employing a novel mouse model utilizing doxycycline-responsive expression of eIF4E localized to specific phases of hematopoietic maturation. A paradigm example of ribosomopathies, Diamond Blackfan Anemia (DBA) results in erythroid aplasia and failure to respond to erythropoietin (Epo), a key signal in differentiation of early erythroid precursors. However, why hematopoietic precursors in DBA fail to generate the appropriate nascent gene expression response to these cues is still poorly understood. Aim 2 will use employ a universally-applicable, mass spectrometry-based method (OPP-ID) pioneered by Dr. Forester to characterize the nascent proteomic landscape in healthy and DBA-derived iPSCs in response to erythropoietin. Identification of impaired proteomic responses in DBA iPSCs will give insight into how specific ribosomal mutations prevent translation of key genes crucial in early differentiation. This technology will be applied to understand how dexamethasone, a known mediator of hematopoietic rescue of unclear mechanism, remodels the erythroid proteome in response to Epo. Successful completion will result in both a unique contribution into the fields of translational control and erythropoiesis as well as a novel approach to understanding how the nascent translatome is impaired in ribosomopathies. Accomplishment of this proposal is crucial in Dr. Forester’s long-term goals of becoming an independently funded researcher and an expert in the field of proteomic networks governing early hematopoiesis.

科学摘要 红细胞生成是由触发基因网络表达的信号线索的复杂相互作用引导的 产生成熟红细胞所必需的。红系前体细胞对这些信号的快速反应 暂时协调红细胞分化所需的功能。及时、适当地选择 mRNA 核糖体的翻译对于维持红细胞前体之间的平衡至关重要 差异化。这个概念被称为翻译控制，它控制着 mRNA 翻译的效率，因此 在调节对贫血和缺氧等细胞外信号的反应中起着至关重要的作用。失败的 调节这一过程的核糖体机制是核糖体病的基础，核糖体病是由一系列人类疾病引起的 核糖体突变对造血成熟产生深远影响。这个提议 重点关注翻译控制如何在正常情况下指导 mRNA 选择的突出基本问题 红细胞生成和 2.) 在核糖病中响应信号线索而受损。目标 1 将探索如何 eIF4E 是翻译起始的一个关键因素，它以动态的、细胞固有的方式被抑制，以直接翻译 一组特定的转录本。未能抑制成熟红细胞中的 eIF4E 活性会导致不适当的 维持早期红细胞前体和分化受损所需的基因表达。这 target 将机械地破译目标 mRNA 中的基序，并破译 eIF4E 如何特异性识别这些基序 核糖体募集的转录本。将测试 eIF4E 严格调节对红细胞生成的依赖性 体内采用一种新型小鼠模型，利用多西环素响应性表达 eIF4E 定位于特定区域 造血成熟阶段。核糖体病的典型例子，钻石黑扇贫血症 (DBA) 导致红细胞再生障碍和对促红细胞生成素 (Epo) 没有反应，促红细胞生成素是早期红细胞分化的关键信号。 红细胞前体。然而，为什么DBA中的造血前体细胞无法生成适当的新生细胞？ 基因表达对这些线索的反应仍然知之甚少。目标 2 将使用普遍适用的、 Forester 博士首创的基于质谱的方法 (OPP-ID)，用于表征新生蛋白质组学 健康和 DBA 衍生的 iPSC 对促红细胞生成素的反应。受损蛋白质组的鉴定 DBA iPSC 中的反应将深入了解特定核糖体突变如何阻止关键基因的翻译 对早期分化至关重要。该技术将用于了解地塞米松（一种已知的药物）如何 机制尚不清楚的造血拯救介质，响应 EPO 重塑红细胞蛋白质组。 成功完成将为翻译控制和 红细胞生成以及一种了解新生翻译组如何受损的新方法 核糖病。该提案的完成对于 Forester 博士成为一名 独立资助的研究人员和控制早期造血的蛋白质组网络领域的专家。