Cap-Dependency in Hematopoietic Stem and Progenitor Cell Translation

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Project Abstract Hematopoietic stem and progenitor cells (HSPCs) produce blood and immune cells throughout the lifespan of an organism and must be preserved, especially to survive and function under stresses like infection, inflammation, and bone marrow transplantation. While extensive research has elucidated transcriptional programs and niche factors that maintain HSPCs, few studies have explored post-transcriptional mechanisms, which are critical for rapid stress responses. Translation regulation allows cells to quickly shift translational output in response to stimuli, including stressors and growth cues, by targeting single or groups of mRNAs based on unique or shared translation regulatory motifs. Stem cells are known to have characteristically low rates of total protein synthesis in comparison to differentiated cells; however, few studies have further characterized subtypes of translational regulatory motif activity in HSPCs. My goal is to understand how HSCPs differentially employ mechanisms of translational regulation in steady state and stress. I am particularly interested in regulation of the rate-limiting step of translation: initiation. Most mRNAs require a collection of eukaryotic initiation factors to assemble at the 5’ cap of an mRNA to recruit ribosomes. Terminal oligopyrimidine (TOP) motifs are one example of translation regulatory motifs regulated in this cap-dependent manner; TOPs are pyrimidine-rich sequences that exist in the 5’ UTRs of many growth-associated genes. Alternatively, some mRNAs bypass the need to assemble some or all eukaryotic initiation factors at the 5’ cap and are classified as cap-independent transcripts. Internal ribosome entry sites (IRESs) are one example of a translation regulatory motif regulated in a cap- independent manner. IRESs have been identified in mammalian 5’ UTRs to regulate the translation of genes important for cell survival and differentiation; their unique 5’ UTR secondary structures allow ribosome complexes to load onto mRNAs without some or all of the cap-machinery, especially under stress conditions. In this project, I will compare cap-dependent (TOP) and cap-independent (IRES) translation regulatory motif activity across hematopoiesis in steady state and stress to understand their role in HSPC function and survival. In my first aim, I will create vectors that compare several IRES- and TOP-translation motifs and compare rates of cap-dependent and cap-independent translation across hematopoiesis in steady state. Since translation regulation is particularly relevant to the cellular stress response, my second aim will assess translational changes across hematopoiesis in response to stresses like bacterial infection, chemotherapy, and bone marrow transplantation by assessing reporter activity and performing ribosome-immunoprecipitations to determine changes in the translatome. In my third aim, I will determine if cap-dependent and cap-independent translation rates predict HSPC regeneration of hematopoiesis. Ultimately, this study will glean molecular insight into how protein synthesis is regulated HSCPs, which may offer directions for improving regenerative therapies with stressful ex vivo manipulations like bone marrow transplants.