Understanding tissue selective phenotypes in ribosomopathies with new technologies

在核糖体疾病中，核糖体成分的干扰表达导致组织特异性表型，如 四肢和颅面部缺陷以及骨髓衰竭。是什么导致了这种组织选择性 核糖体是一种普遍存在的细胞机器，由于核糖体突变而产生的表现仍然是 神秘感。我们的初步数据有力地支持了翻译功能障碍可能导致疾病 发病机制。特别是，我们的发现表明，翻译对基因表达的特异性在RP上 单倍体功能不全可能源于一条中间通路，即P53-4E-BP1-eIF4E轴，它成为 激活的RP单倍体不足与帽依赖翻译的选择性变化有关，即mRNAs 具有需要eIF4A解旋酶活性的结构化5‘UTRs。这一初步数据有力地支持了 开发既能检查翻译控制又能检查蛋白质合成的技术 以前无法解决的造血室，限制了我们的 对DBA发病机制的认识。引人注目的是，虽然人们已经知道RP突变已经有20多年了 导致与核糖体疾病相关的骨髓衰竭，目前还没有任何全基因组研究 精确定位体内血液学异常潜在的特定翻译损伤。这至少是部分原因 由于能够使用诸如小分子核糖体图谱分析等技术方面的技术限制 单元格数量。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，体内5‘UTRRNA结构分析使用新的 我们已经试验的称为细胞内突变和图谱(IcM2)的技术，以及 蛋白质合成。这直接回应了由该RFA发起的工具和技术开发目标。 在目标1中，我们将利用一种针对小细胞数量进行优化的新技术来表征翻译 第一次在信徒的造血室中进行基因表达 核糖体病小鼠模型。这将使描述基因的全球翻译图景成为可能 首次在核糖病的体内表达与造血功能障碍有关。在目标2中，我们将 开发一种我们开创的已知icM2的新技术，以解决 选择性mRNAs的5‘UTRs中是否存在共同的结构特征，这可以解释特异性 核糖体疾病背后的基因表达变化。这项技术具有广泛的影响。 因为它将允许史无前例地解析任何细胞类型的RNA结构。总而言之，这项提案 将开发最先进的技术，这有可能改变我们对整个 人类疾病的类别。

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.