Understanding tissue selective phenotypes in ribosomopathies with new technologies

在核糖体病中，核糖体组分的表达受到干扰导致组织特异性表型，如 如四肢和颅面缺陷以及骨髓衰竭。是什么导致了这种组织选择性 由于核糖体（一种无处不在的细胞机器）突变而引起的表现， 谜我们的初步数据强烈支持翻译功能障碍可能有助于疾病 发病机制特别是，我们的研究结果表明，翻译特异性基因表达后RP 单倍不足可能是由中间途径p53- 4 E-BP 1-eIF 4 E轴引起的， 激活，并将RP单倍不足与帽依赖性翻译的选择性变化，即mRNA 具有需要eIF 4A解旋酶活性的结构化5 'UTR。这一初步数据有力地支持了 开发既可以检查翻译控制又可以检查细胞内蛋白质合成的技术。 造血隔室，以前无法解决，并限制了我们的 了解DBA的发病机制。引人注目的是，虽然20多年来人们已经知道RP突变 导致与核糖体病相关的骨髓衰竭，还没有任何全基因组研究， 精确定位体内血液学异常背后的特异性翻译损伤。这至少部分是 由于在能够使用诸如核糖体谱分析的技术方面的技术限制， 细胞的数量。我们建议通过开发新技术和最先进的 方法包括低输入核糖体分析，使用新的 我们已经试验了称为细胞内突变和映射（icM 2）的技术， 蛋白质合成这直接回应了RFA发起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量优化的新技术来表征转译细胞。 基因表达的景观第一次在一个忠实的造血隔室 核糖体病小鼠模型。这将使得能够表征基因的全局翻译景观， 首次在核糖体病中发现了体内造血功能障碍的潜在表达。在目标2中，我们 开发一种新的技术，我们已经率先已知的icM 2，以解决的基本问题， 选择性mRNA的5 'UTR中是否存在共同的结构特征， 核糖体病的基因表达改变。这项技术具有广泛的影响 因为它将允许前所未有的分辨率从任何细胞类型的RNA结构。总之，这一提议 我们将开发最先进的技术，这些技术有可能改变我们对整个地球的理解。 类人类疾病。

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.