Ribosomal protein mutations in genes such as rps19 and rpL11 cause blood specific defects, and this is seen in patients with DBA. We described erythroid and HSC defects in the zebrafish embryo resulting from homozygous mutation of rps29, a protein in the 40s small subunit of the ribosome. We identified a gene expression signature of p53 activation in the rps29 mutant and tested the role of p53 activation genetically by generating rps29-/-;p53-/- embryos. Loss of p53 rescues all of the hematopoietic and morphological phenotypes in the rps29 mutant that we tested. Skeletal and neuronal tissues are also affected, including a gene, otpa, whose expression is dependent on rps29 expression level.
Our work builds upon the previously characterized HSC and artery defects of the rps29 mutant[20
]. We went on to characterize red blood cells in the mutant, as ribosomal protein mutations cause erythroid specific defects in patients with DBA. (N.B. Mutations in rps29 have not been found in DBA patients.) Red blood cells in the homozygous rps29 mutant are indeed affected as hemoglobin levels are decreased. Since embryonic globin RNA is normal, this defect may be at a terminal stage of differentiation or specific to hemoglobinization. This phenotype is in contrast with the erythroid progenitor block seen in DBA patients, but mirrors what is seen in the zebrafish rps19 morphant and rpL11 mutant. Although the specific stage of the erythropoietic defect differs in zebrafish ribosomal protein mutants and patients, the trends of the zebrafish mutants phenocopy DBA in both skeletal and hematopoietic tissues. Erythroid cells are affected, but myeloid tissues remain largely unaffected in both cases. Mechanistically, the role of p53 activation in the rps29 mutant is consistent with other ribosomal protein mutants and DBA models.
Our data demonstrate that p53 is a mediator of the rps29 mutant phenotype. Previous studies have suggested a critical role for p53 in ribosomal protein mutant phenotypes, and our findings extend this observation to an additional ribosomal protein, rps29. GSEA shows strong enrichment for a p53 activation gene signature, as 22 out of 31 genes in a p53-dependent irradiation signature are also up in the rps29 mutant. Studies from others suggest that the mechanism of p53 activation is nucleolar/ribosomal stress[13
]. In this model, a mutated ribosomal protein prevents ribosomal subunit formation, leading to an increase in free ribosomal proteins. Some of these, including rpL11 and rpL5, can bind mdm2 and sequester it from p53, leading to p53 activation. Our data suggest that the mechanism of p53 activation in the mutant mirrors that of irradiation, the source of p53 activation in our analysis. Interestingly, there is evidence that some DNA damage agents can initiate ribosomal stress[34
], and a common pathway could explain the similar gene signatures. However, the downstream targets of p53 in the rps29 mutant are not yet fully elucidated.
It is interesting that p53 knockdown also rescues the hemoglobin defect, as this suggests that p53 activation is causing the hemoglobin defect. Other zebrafish studies have tested the role of p53 activation with conflicting results; a p53 morpholino rescues the hemoglobin defect in the rpL11 mutant, but does not rescue the defect induced by rps19 morpholino knockdown[16
]. Interestingly, morpholino knockdown of telomerase reverse transcriptase (TERT) in zebrafish embryos also causes apoptosis and hematopoietic specific defects[29
]. Like the rps29 mutant, many of the zTERT morphant phenotypes are p53-dependent; in contrast, the zTERT hemoglobin defect cannot be rescued by p53 knockdown. The reason for these discrepancies is unclear, but could be the result of differences between mutants and morpholino knockdown. Homozygous embryos can get maternally deposited RNA from a heterozygous parent, whereas an ATG morpholino would inhibit translation of maternal RNAs and cause a more severe knockdown, making rescue more difficult. We still do not know how p53 activation inhibits heme biosynthesis. One hypothesis is based on evidence that p53 in the mitochondria is involved in heme biosysthesis and iron-sulfur (Fe-S) cluster formation. Heme biosynthesis requires synthesis of ALAS2, which our lab has previously shown to be inhibited when Fe-S cluster biogenesis is impacted[30
]. Assembly of Fe-S clusters requires intact mitochondria[35
], but p53 activation causes mitochondrial membrane permeabilization[36
]. In this model, p53 activation would affect Fe-S cluster biogenesis and subsequent heme biosynthesis, and the effect would be rescued by p53 knockdown.
An advantage of using a zebrafish mutant for these studies is the ability to compare both heterozygous and homozygous mutants, and subsequently identify gene expression patterns that correlate with rps29 level. Although we could not separate wildtype and heterozygous embryos for the microarray, we could still identify dosage specific defects by whole mount ISH. Orthopedia protein a (otpa
) is a homeobox containing gene expressed in the brain and important for neuron development[31
], and the levels of otpa
depend on rps29
levels. The existence of dose-dependent genes with respect to a ribosomal protein suggests a dosage sensitivity model. In wildtype embryos, the ribosome can function normally, translation occurs normally, and genes such as myb, flk1
, and otpa
are expressed as expected. In the homozygous rps29 mutant, there is a downstream effect on myb, flk1
, and otpa
expression. When only one copy of rps29 is mutated, only otpa
is down-regulated and many other genes are unaffected. An alternate hypothesis is that otpa
expression is not differentially regulated, but stability of otpa
mRNA is affected by rps29 mutation. Regulation of RNA expression or stability could still be dose-dependent on rps29 mRNA levels. Interestingly, there are cases where relatives of DBA patients have the same mutation but milder symptoms of the disease, and this may be the result of different expression levels of the functional ribosomal protein. Our data are in agreement with the hypothesis that differences of a ribosomal protein level could cause a change in disease presentation.
p53 mutation partially rescues otpa in the homozygous mutant to heterozygous levels, suggesting that p53 activation may play a role in both the heterozygous and homozygous rps29 phenotype. The fact that p53 mutation only partially rescues otpa mRNA levels further suggests that otpa is affected by rps29 knockdown by both p53-dependent and p53-independent mechanisms. It is possible that a protein not efficiently translated in the rps29 heterozygote (or homozygote) directly regulates otp transcription, leading to a dose-dependent decrease in otpa mRNA expression in the rps29 mutant. Another possibility is that otpa directly interacts with the ribosome. There may also be a p53-independent stress signal in the rps29 heterozygote and homozygote, causing some neuronal tissues to die without p53 activation. Further testing is required to determine the exact p53-independent mechanism causing a decrease of otpa mRNA in the rps29 mutant.
There are still lingering questions about the exact mechanism downstream of ribosomal protein mutation, and modifier screens in the zebrafish could address these questions. The zebrafish embryo is amenable to in vivo chemical and genetic screens. For example, a genetic screen for rescue of the rps29 mutant would identify novel pathways, p53 dependent or independent, mediating the mutant phenotype. These pathways can provide insight into exactly how erythropoiesis and HSC formation are affected by ribosomal protein mutation and p53 activation. An in vivo chemical screen for compounds that rescue the hematopoietic defect would identify therapeutics that may be more likely to translate to patients than hits from an in vitro screen. The unique advantages of a zebrafish DBA model and ribosomal deficiencies hold promise for the field of ribosomal protein biology.