Advances in understanding the control of gene expression by epigenetic, genetic, and transcriptional mechanisms have identified key regulators of organismal development and differentiation, with critical implications for the diagnosis and treatment of disease. In contrast, regulators of mRNA translation remain poorly understood. Recent findings are shifting the view of translation from one effected by static `protein factories' to one with an unexpected role regulating responses to differentiation signals and stress. These novel findings are highlighted by the discovery of ribosomopathies, diseases that stem from abnormalities of a universal cellular organelle, the ribosome, but show effects limited to specific tissues. Diamond Blackfan anemia (DBA), an inherited bone marrow failure syndrome characterized by severe anemia, congenital malformations, and predisposition to cancer, is one such ribosomopathy, where alterations in one of 16 ribosomal protein constituents lead to impaired ribosome assembly and reductions in ribosomes available for protein translation. Despite progress defining its genetic basis, there is little understanding of how such global ribosomal defects cause the features of DBA. Establishing why severe anemia results from specific abnormalities in this fundamental cellular constituent is likely to be critical to the development of improved treatment approaches for DBA. Similarly, understanding this mechanism may highlight shared sensitivities with other target tissues, with broad implications in the study of birth defects and neoplasia. Furthermore, understanding of the connections between these specific ribosomal abnormalities and limited tissue pleiotropy in DBA will serve as a model for the study of other ribosomopathies where diverse disorders include both hematologic and non-hematologic disease. A confluence of novel methods including cell culture techniques permitting expansion of human blood progenitor cells into red cell precursors, technical innovations that allow genome-wide measurement of protein translation from mRNA, and systems-based computational methods for data analysis make questions about mRNA translation in patient samples amenable to analysis at whole translatome scale. In this work the Project Leader will employ sequencing of ribosome-associated RNA transcripts to identify the changes in protein translation that characterize the normal process of differentiation from a multipotent blood cell progenitor into committed red cell precursor. From this baseline, the Project Leader will compare polysome-associated mRNAs in DBA to define and validate abnormalities in the control of protein synthesis by red cell progenitors in patients of multiple genotypes to identify the critical translational pathways underlying DBA. Finally the Project Leader will use precise proteomic assessment of normal and DBA ribosomes to address the question of whether the protein synthesis apparatus is qualitatively as well as quantitatively different in DBA patients.
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