Understanding tissue selective phenotypes in ribosomopathies with new technologies

利用新技术了解核糖体病的组织选择性表型

• 批准号: 10506560
• 负责人: Maria Barna
• 金额: $ 23.91万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10506560
• 关键词: 5' Untranslated Regions; Address; Apoptosis; Biogenesis; Bone marrow failure; Cell Count; Cell Cycle Arrest; Cells; Congenital Abnormality; Craniofacial Abnormalities; Data; Development; Diamond-Blackfan anemia; Digit structure; Disease; EIF4EBP1 gene; Elements; Erythroid; Failure; Functional disorder; Gene Expression; Genes; Genetic Models; Global Change; Goals; Hematology; Hematopoietic; Human; Impairment; Knowledge; Lead; Limb structure; Link; Maps; Measurement; Mediating; Messenger RNA; Methodology; Molecular; Monitor; Mutate; Mutation; Pathogenesis; Pathologic; Pathway interactions; Phenotype; Population; Protein Biosynthesis; RNA; Regulation; Resolution; Ribosomal Proteins; Ribosomes; Specificity; Stress; Structure; Syndrome; TP53 gene; Technology; Time; Tissues; Transcript; Translating; Translations; cell type; chromosome 5q loss; craniofacial; disease phenotype; erythroid differentiation; experimental study; genome-wide analysis; helicase; hematopoietic differentiation; human disease; in vivo; mouse model; new technology; novel; ribosome profiling; technology development; tool development; transcription factor

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes, such as limb and craniofacial defects as well as bone marrow failure. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Our preliminary data strongly support that translational dysfunction may contribute to disease pathogenesis. In particular, our findings show that translational specificity to gene expression upon RP haploinsufficiency may arise from an intermediary pathway, the p53-4E-BP1-eIF4E axis, which becomes activated and links RP haploinsufficiency to selective changes in cap-dependent translation, namely mRNAs with structured 5’UTRs that require eIF4A helicase activity. This preliminary data strongly supports the development of technologies that can both examine translational control and protein synthesis within the hematopoietic compartment that have been previously unattainable to resolve and have limited our understanding of DBA pathogenesis. Strikingly, while it has been known for over 20 years that RP mutations lead to bone marrow failure associated with ribosomopathies, there has not been any genome-wide studies to pinpoint specific translation impairments underlying hematological abnormalities in-vivo. This is at least in part due to a technical limitation in being able to employ technologies such as ribosome profiling analysis for small numbers of cells. We propose to close this gap by developing new technologies and state-of-the-art approaches including low input ribosome profiling, in-vivo 5’UTR RNA structure analysis using a new technology that we have piloted known as in-cell mutate and map (icM2), and single cell measurements of protein synthesis. This directly answers to the tool and technology development goals sponsored by this RFA. In Aim 1, we will utilize a new technology optimized for small cell numbers to characterize the translational landscape of gene expression for the first time within the hematopoietic compartment of a faithful ribosomopathy mouse model. This will enable characterization of the global translation landscape of gene expression underlying hematopoietic dysfunction in vivo in ribosomopathies for the first time. In Aim 2, we will develop a new technology that we have pioneered known icM2 to address the fundamental question of whether shared structural features are present in the 5’UTRs of selective mRNAs that account for specificity to gene expression changes underlying ribosomopathies. This technology has broad reaching implications because it will allow unprecedented resolution of RNA structures from any cell type. Together, this proposal will develop state-of-the art technologies, which holds the potential to transform our understanding of an entire class of human diseases.

在核糖体病中，核糖体成分的扰动表达会导致组织特异性表型，这种表型 作为肢体和颅面缺陷以及骨髓衰竭。什么是这种组织选择性的 核糖体突变（一种无处不在的细胞机）的表现一直保持不变 神秘。我们的初步数据强烈支持转化功能障碍可能导致疾病 发病。特别是，我们的发现表明RP对基因表达的转化特异性 单倍不足可能来自中间途径，即p53-4e-bp1-eif4e轴，它变成 激活的并将RP单倍度弥补与cap依赖性翻译的选择性变化，即mRNA 具有需要EIF4A解旋酶活性的结构化5'UTR。此初步数据强烈支持 可以开发可以检查转化控制和蛋白质合成的技术 造血室以前无法解决并限制了我们的 了解DBA发病机理。令人惊讶的是，虽然已知20多年了RP突变 导致骨髓衰竭与核糖瘤相关，尚未进行任何基因组的研究 查明体内血液学异常的特定翻译障碍。至少部分是 由于技术限制，能够员工技术，例如小型核糖体分析分析 细胞数量。我们建议通过开发新技术和最新技术来缩小这一差距 使用新的方法，包括低输入核糖体分析，体内5'UTR RNA结构分析 我们试行的技术被称为细胞内突变和MAP（ICM2），以及单细胞测量 蛋白质合成。这直接回答了该RFA赞助的工具和技术开发目标。 在AIM 1中，我们将利用针对小单元数进行优化的新技术来表征翻译 基因表达的景观首次在忠实的造血区内 核糖瘤病小鼠模型。这将使基因的全球翻译格局能够表征 首次在核糖体病中体内造血功能障碍的表达。在AIM 2中，我们将 开发了一种新技术，我们已经开创了已知的ICM2，以解决的基本问题 选择性mRNA的5'Utrs中是否存在共享的结构特征，以说明特异性 基因表达会改变核糖体病。这项技术具有广泛的影响 因为它将允许从任何细胞类型的RNA结构进行前所未有的分辨率。在一起，这个建议 将开发最先进的技术，这有可能改变我们对整个的理解 人类疾病类。