EpoR & Stat5 regulation of ribosome biogenesis and protein synthesis in erythropoiesis

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Project Summary Protein synthesis and ribosome biogenesis are the two most energy -intensive processes in a cell, resulting in their regulation through tight controls. EpoR, a member of the cytokine receptor superfamily, and its downstream mediator Stat5, are essential for erythropoiesis. The current proposal addresses novel regulation of ribosome biogenesis and protein synthesis by EpoR and Stat5 signaling during a key erythroid developmental decision. In recently published work we found that EpoR/ Stat5 stimulate shorter and more numerous cycles in early erythroblasts, while also promoting formation of larger erythroblasts that mature into larger red cells, in both mice and humans. This surprising finding suggests that EpoR/ Stat5 signaling alters the relationship between cell cycle duration and cell size, simultaneously inducing shorter cycles and exceptionally fast growth in biomass. In preliminary data supporting this hypothesis, single-cell RNA sequencing of Epor-/- and Stat5-/- fetal livers show dysregulated expression of ribosome biogenesis and translation genes. Further, we identified an EpoR/ Stat5-dependent sharp spike in the rate of rRNA transcription (~2.5 fold), protein synthesis rate (~4 to 6 fold) and rate of growth in cell size (~3 fold), that coincides with a key cell fate decision. It takes place as early erythroid progenitors known as CFU-e transition from self-renewal to erythroid terminal differentiation (ETD), becoming erythroblasts. The spike in protein synthesis at this time also coincides with an unusually short cell cycle. Immediately following the CFU-e/ETD transition, the rates of ribosome biogenesis and protein synthesis begin to decline back to baseline together with the decline in cell cycle speed, even though erythroblasts continue to divide and synthesize hemoglobin for an additional 3 to 5 cell divisions. The coincidence in the spikes of protein synthesis and ribosome biogenesis with cell cycle shortening at the CFU-e/ETD switch suggests that these processes are linked and may be functionally relevant to the switch. We will investigate the EpoR/Stat5- induced spike in protein synthesis with the following aims: Aim 1: Investigate the EpoR /Stat5 -induced spike in ribosome biogenesis & protein synthesis, determining the intracellular signaling pathways that are mediating this spike and identifying a potential subset of transcripts whose translation rate spikes. Aim 2: Determine causal relationships between the cell cycle, ribosome biogenesis, protein synthesis and cell size. The regulatory interactions between cell cycle duration, protein synthesis rate and cell size in mammalian cells are not well understood and yet are critical in development and in cancer. Aim 3: Test the hypothesis that the EpoR /Stat5 -induced spike in protein synthesis is required for erythroid differentiation. We will determine whether the EpoR-induced spike in protein synthesis is an 'Achilles heel' in mice deficient in the ribosomal protein Rpl11, a model of Diamond Blackfan Anemia, potentially explaining the selective sensitivity of the erythroid lineage to ribosomopathies.