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的翻译控制和新生蛋白质组反应

• 批准号: 9901525
• 负责人: CRAIG M. FORESTER
• 金额: $ 16.85万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9901525
• 关键词: Anemia; Biological Process; CD34 gene; Cells; Complex; Coupled; Cues; Defect; Dependence; Dexamethasone; Diamond-Blackfan anemia; Disease; Doxycycline; Elements; Epigenetic Process; Equilibrium; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Erythropoietin; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factors; Event; Exposure to; Failure; Funding; Gene Expression; Genes; Genetic; Genetic Transcription; Genetic Translation; Goals; Hematopoiesis; Hematopoietic; Hour; Hypoxia; Impairment; Label; Lead; Life; Light; Maintenance; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Messenger RNA; Methods; Modeling; Mutation; Outcome; Patients; Pharmaceutical Preparations; Pharmacology; Phase; Play; Post-Transcriptional Regulation; Process; Production; Protein Biosynthesis; Proteins; Proteome; Proteomics; RNA Cap-Binding Proteins; Regulation; Regulatory Element; Repression; Research; Research Personnel; Ribosomal Proteins; Ribosomes; Role; Signal Transduction; Specificity; Stimulus; Techniques; Technology; Time; Transcript; Transgenic Mice; Translating; Translation Initiation; Translations; Umbilical Cord Blood; base; erythroid differentiation; extracellular; human disease; human model; in vivo; in vivo evaluation; induced pluripotent stem cell; insight; mouse model; novel; novel strategies; novel therapeutic intervention; overexpression; prevent; programs; recruit; response; stem; transcriptome sequencing; translatome

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.

科学抽象 红细胞生成受触发基因网络表达的信号线索的复杂相互作用的指导 这是生产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 在时间上协调红细胞分化所需的功能。及时和适当地选择mRNA 对于维持红细胞前体之间的平衡至关重要 分化这个概念被称为翻译控制，它控制着mRNA翻译的效率， 在调节对细胞外信号如贫血和缺氧的反应中起关键作用。失败 核糖体机制来调节这一过程的基础核糖体病，一组人类疾病， 从核糖体突变导致造血成熟的深远后果。这项建议 集中在如何翻译控制的突出的，基本的问题1。在正常细胞中指导mRNA选择 红细胞生成和2.）在核糖体病中对信号线索的反应受损。目标1将探讨如何 eIF 4 E是翻译起始中的关键因子，它以动态的细胞内在方式被抑制，以指导 一组特定的记录未能抑制成熟成红细胞中的eIF 4 E活性导致不适当的 维持早期红细胞前体和受损分化所必需的基因表达。这 aim将机械地破译靶mRNA中的基序，并破译eIF 4 E如何特异性地识别这些基序。 核糖体募集的转录物。将测试eIF 4 E的紧密调节对红细胞生成的依赖性 在体内，使用新的小鼠模型，利用定位于特异性的eIF 4 E的多西环素应答性表达， 造血成熟阶段。核糖体病的典型例子，钻石布莱克凡贫血（DBA） 导致红细胞发育不全和对促红细胞生成素（Epo）的反应失败，促红细胞生成素是早期分化的关键信号。 红细胞前体然而，为什么DBA中的造血前体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将使用一个普遍适用的， 基于质谱的方法（OPP-ID）由Forester博士开创，用于表征新生蛋白质组 健康和DBA衍生的iPSC对促红细胞生成素的反应。受损蛋白质组学鉴定 DBA iPSC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了解地塞米松，一种已知的 机制尚不清楚的造血拯救介质，响应Epo重塑红系蛋白质组。 成功完成将导致对翻译控制领域的独特贡献， 红细胞生成以及一种新的方法来了解新生翻译组是如何受损， 核糖体病实现这一建议是至关重要的博士福雷斯特的长期目标成为一个 独立资助的研究人员和蛋白质组学网络管理早期造血领域的专家。