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.