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

科学摘要 红细胞生成是由触发基因网络表达的信号信号复杂相互作用所引导的 是生产成熟红细胞所必需的。红系前体对这些信号的快速反应 暂时协调红系分化所需的功能。及时和适当地选择mRNAs 核糖体的翻译对于维持红系前体细胞之间的平衡是必不可少的 差异化。这一概念被称为翻译控制，它支配着mRNA翻译的效率，因此 在调节对细胞外信号的反应中起着至关重要的作用，如贫血和缺氧。失灵了 核糖体调节这一过程的机制是核糖体疾病的基础，核糖体疾病是一系列人类疾病的结果 从核糖体突变导致对造血成熟的深远影响。这项建议 专注于如何平移控制的突出的基本问题1。)在正常情况下指导mRNA选择 红细胞生成和2)在核糖体疾病中对信号提示的反应受损。目标1将探讨如何 EIF4E，翻译启动中的一个关键因素，以一种动态的、细胞固有的方式被抑制，以指导 一组特定的成绩单。未能抑制成熟红细胞中eIF4E活性导致不适当 维持早期红系前体细胞和受损分化所必需的基因表达。这 AIM将机械地破译目标mRNA中的基序，并破译eIF4E如何具体识别这些基序 核糖体招募的转录。EIF4E的严格调控对红细胞生成的依赖性将被测试 体内利用多西环素反应性表达特异性eIF4E的小鼠模型 造血成熟的各个阶段。核糖体病的一个范例，钻石布莱克凡贫血(DBA) 导致红系再生障碍性贫血和对促红细胞生成素(EPO)的反应失败，EPO是早期白血病分化的关键信号 红系前体。然而，为什么DBA中的造血祖细胞不能产生适当的新生 对这些线索的基因表达反应仍然知之甚少。目标2将使用普遍适用的、 基于质谱学的方法(OPP-ID)由Forester博士首创，用于表征新生蛋白质组 健康和DBA来源的IPSCs对促红细胞生成素的反应。受损蛋白质组的鉴定 DBA ipscs的反应将深入了解特定的核糖体突变如何阻止关键基因的翻译 在早期分化中至关重要。这项技术将被应用于理解地塞米松，一种已知的 机制不明的造血挽救介质，重建红细胞蛋白质组以响应EPO。 成功的完成将导致翻译控制领域的独特贡献和 红细胞生成以及一种新的理解新生翻译组如何受损的新方法 核糖体疾病。完成这项提议对福雷斯特博士成为一名 独立资助的研究员和管理早期造血的蛋白质组网络领域的专家。

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.