Nucleolar Stress and p53 Mediated Erythroid Failure in Diamond Blackfan Anemia

The discovery that several ribosomal protein genes can be mutated in DBA suggests that ribosomal protein gene mutations may account for many or all cases of DBA, and focuses attention on the ribosome. While experiments in yeast and mammalian cells show that RPS19 depletion or mutation leads to a block in ribosomal RNA biosynthesis, this result does not explain why erythropoiesis is so severely affected in DBA. We hypothesize that during fetal development immature erythroid cells proliferate more rapidly than other lineages and therefore require very high ribosome synthetic rates to generate sufficient capacity for translation of erythroid specific transcripts that must take place before these unique cells enucleate. To test this kinetic hypothesis we measured RNA biogenesis in primary mouse fetal liver cells and reported previously that during the first 24 hours cell number increases 3–4 fold while, remarkably, there is a 6-fold increase in RNA content during the same period, suggesting that the cells accumulate an excess of ribosomal RNA (80% of measured RNA) during early erythropoiesis. Retrovirus infected siRNA RPS19 knockdown cells show reduced proliferation of FACS sorted GFP positive cells at 48 hours. Although the cell yield is reduced, the differentiation pattern of the surviving GFP positive cells is similar to that of the controls. While quantitative RT-PCR analysis shows that RPS19 mRNA is rapidly depleted, Western analysis during this time course does not show a deficiency of RPS19 protein. This suggests strongly that the proliferative defect is not due to insufficiency of RPS19 protein, and is more likely due nucleolar stress induced by the block in ribosome biogenesis. Molecular consequences could lead to redistribution of cell cycle proteins normally resident in the nucleolus with consequent p53 mediated cell cycle arrest and or apoptosis. To test this hypothesis we used a culture system that allows expansion without differentiation of immature cells in SCF, EPO, IGF-1 and dexamethasone. Under these conditions proliferation of siRNA expressing precursors is reduced with an increased proportion arrested in G0/G1 in the knockdown cells. Furthermore, p53 is increased in the knockdown cells. Taken together, these data suggest that RPS19 insufficient cells undergo a nucleolar stress response and erythroid cells proliferate poorly because of p53 mediated cell cycle arrest and apoptosis.