Figure 1.
Protein haploinsufficiency and polysome defects in Rps19 and Rpl5 mutant mouse embryonic stem cells.
For immunoblotting, ES cell lysates were separated using gel electrophoresis, transferred to a nitrocellulose membrane, and blotted with antibodies against RPS19 and RPL5. β-Actin was used as a loading control. Rps19 mutant (A) and Rpl5 mutant (C) ES cells showed protein haploinsufficiency (upper panels); β-Actin confirmed similar protein loading for mutant and parent (lower panels). For analyses of polysome profiles, ES cells were incubated in the presence of cycloheximide, lysed, and layered onto sucrose gradients. After ultra-centrifugation, polysome profiles were retrieved using an ISCO density gradient fractionator and UA-6 UV/Vis detector. RPS19 haploinsufficient ES cells (B, lower panel) showed a decreased 40S peak when compared to the parental line (B, top panel). In contrast, RPL5 haploinsufficient cells (D, lower panel) had a decreased 60S subunit compared with the parental cells (D, top panel).
Figure 2.
Embryoid body (EB) formation is impaired in both Rps19 and Rpl5 mutants.
ES cells were differentiated into EBs and scored on day 4 to assess total number of EBs formed. Both mutants showed a reduction in EB formation when compared to the parental cells (3 independent experiments).
Figure 3.
Primitive erythropoiesis is defective in Rps19 and Rpl5 mutants.
Day 4–5 EBs were harvested, made into single cell suspension, and added to primitive erythroid differentiation media. Colonies were scored on day 7. Both Rps19 mutant (A) and Rpl5 mutant (B) cell lines exhibited a severe defect in primitive erythroid colony formation. (Rpl5-5 independent pooled experiments, Rps19-3 independent pooled experiments).
Figure 4.
The differentiation defects observed in Rps19 and Rpl5 mutants are nonspecifically rescued by p53 inhibition.
(A) Western blot analyses were performed from mutant ES cells with antibodies against p53, using β-Actin as a loading control. ES cells from the Rps19 mutant cells showed an increase in p53 expression. In contrast, the Rpl5 mutant expressed no increase in p53, compared with the parental line. Image J quantification of western blots from 3 independent experiments demonstrated that the Rps19 mutant ES cells had approximately a 4-fold increase in p53 protein compared to the wild type cells. (B) qRT-PCR performed on these ES cells showed an increase in p21 mRNA only in the Rps19 mutant ES cells (3 independent experiments) while there was no similar increase in the Rpl5 mutant ES cells (4 independent experiments). siRNA targeting p53 was used to transiently transfect ES cells 24 hours prior to primary differentiation, obtaining >90% p53 knockdown by qRT-PCR. Both mutants (C) showed a significant increase in EB formation with p53 knockdown (4 independent pooled experiments). This effect was nonspecific, as p53 knockdown of parental cells also increased EB formation (D). The primitive erythroid colony defect was partially compensated in the Rps19 mutant after p53 inhibition and overcompensated in the Rpl5 mutant (E) (3 independent pooled experiments). This augmentation of colony formation was again nonspecific, as there was an increase in primitive colony formation with p53 knockdown in both parental ES cells when compared with the control siRNA (3 independent pooled experiments for Rpl5 parent and 4 independent experiments for Rps19 parent) (F).
Figure 5.
Rpl5 mutant ES cells exhibit a p53-independent cell cycle arrest.
Cell cycle analyses were performed by fixing ES cells with 70% ethanol, followed by staining with PI solution containing RNase A. Quantification of cell cycle phases (A), along with flow cytometry profiles (B) of Rps19 mutant ES cells show no difference, compared to the parent. In contrast, the cell cycle profile of the Rpl5 mutant ES cells exhibited a three-fold increase in the G2 phase with a concomitant decrease in the G1 and S phases, consistent with a delayed G2 phase transition (A, C) (three independent pooled experiments). Stable transfection of the Rpl5 mutant with a vector containing Rpl5 cDNA showed complete correction of the cell cycle defect; however, siRNA knockdown of p53 was unable to rescue the defect (D).
Figure 6.
Rpl5 mutant ES cells exhibit more severe growth defect than Rps19 mutant cells.
Cells were seeded in 6 well plates at a concentration of 5×103 per well with ES maintenance media, and live cell counts were performed daily for 5 days using Trypan blue. The total number of cells from the two mutants were normalized to their respective parental line and represented as a percentage. From days 3–5 of culture, the Rpl5 mutant ES cells expanded at a significantly slower rate, when compared with the Rps19 mutant ES cells (three independent pooled experiments).
Figure 7.
Proposed model suggesting a secondary role for p53 in augmenting erythroid colony formation in mouse ES cell models of Diamond Blackfan anemia.
Wild type mouse embryonic stem (ES) cells can be differentiated into primitive erythroid colonies (A). In the normal setting, colony formation can be further increased by p53 knockdown. (B) Rps19 mutant ES cells exhibit defective primitive erythroid colony formation through an unknown p53-independent mechanism. However colony formation can be augmented by p53 knockdown through a separate p53 dependent pathway. (C) The Rpl5 mutant ES cells show an early cell cycle defect at the ES cell stage that is p53-independent. These cells also exhibit a similar defect in primitive erythroid colony formation through a p53- independent mechanism. p53 knockdown in these cells increases colony formation to a greater degree than the Rps19 mutant cells, for unknown reasons.