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

基本信息

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
项目类别：
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&apos 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

项目摘要

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，核糖体成分的表达紊乱会导致组织特异性表型，例如肢体和颅面缺陷以及骨髓衰竭是造成这种组织选择性的原因。核糖体（一种普遍存在的细胞机器）突变导致的表现仍然是一个问题我们的初步数据有力地支持翻译功能障碍可能导致疾病。特别是，我们的研究结果表明 RP 基因表达的翻译特异性。单倍体不足可能源自中间途径，即 p53-4E-BP1-eIF4E 轴，该轴变成激活并将 RP 单倍体不足与帽子依赖性翻译（即 mRNA）的选择性变化联系起来具有需要 eIF4A 解旋酶活性的结构化 5'UTR。该初步数据有力地支持了这一点。开发能够检查翻译控制和蛋白质合成的技术以前无法解决的造血室问题限制了我们的研究对 DBA 发病机制的了解令人震惊，而 20 多年来人们就知道 RP 突变。导致与核糖体病相关的骨髓衰竭，目前还没有任何全基因组研究表明查明体内血液学异常的特定翻译障碍这至少是部分的。由于核糖体谱分析等技术的技术限制我们建议通过开发新技术和最先进的技术来缩小这一差距。方法包括低输入核糖体分析、使用新方法进行体内 5’UTR RNA 结构分析我们已经试点的技术被称为细胞内突变和映射（icM2），以及单细胞测量这直接响应了本 RFA 赞助的工具和技术开发目标。在目标 1 中，我们将利用针对小细胞数优化的新技术来表征翻译首次在忠实的造血室中观察到基因表达的情况核糖体病小鼠模型将能够表征基因的全局翻译景观。在目标 2 中，我们将首次研究核糖体病体内造血功能障碍的表达。开发一项我们首创的新技术 icM2，以解决以下基本问题：选择性 mRNA 的 5'UTR 中是否存在共同的结构特征，从而解释了这项技术具有广泛的影响。因为它将允许对任何细胞类型的 RNA 结构进行前所未有的解析。将开发最先进的技术，这有可能改变我们对整个世界的理解类人类疾病。