Understanding tissue selective phenotypes in ribosomopathies with new technologies

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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.