Deficiency of ribosomal protein S19 during early embryogenesis leads to reduction of erythrocytes in a zebrafish model of Diamond-Blackfan anemia. Hum Mol Genet

Frontier Science Research Center, University of Miyazaki, Miyazaki 889-1692, Japan.
Human Molecular Genetics (Impact Factor: 6.39). 10/2008; 17(20):3204-11. DOI: 10.1093/hmg/ddn216
Source: PubMed


Ribosomes are responsible for protein synthesis in all cells. Ribosomal protein S19 (RPS19) is one of the 79 ribosomal proteins (RPs) in vertebrates. Heterozygous mutations in RPS19 have been identified in 25% of patients with Diamond-Blackfan anemia (DBA), but the relationship between RPS19 mutations and the pure red-cell aplasia of DBA is unclear. In this study, we developed an RPS19-deficient zebrafish by knocking down rps19 using a Morpholino antisense oligo. The RPS19-deficient animals showed a dramatic decrease in blood cells as well as deformities in the head and tail regions at early developmental stages. These phenotypes were rescued by injection of zebrafish rps19 mRNA, but not by injection of rps19 mRNAs with mutations that have been identified in DBA patients. Our results indicate that rps19 is essential for hematopoietic differentiation during early embryogenesis. The effects were specific to rps19, but knocking down the genes for three other RPs, rpl35, rpl35a and rplp2, produced similar phenotypes, suggesting that these genes might have a common function in zebrafish erythropoiesis. The RPS19-deficient zebrafish will provide a valuable tool for investigating the molecular mechanisms of DBA development in humans.

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Available from: Anirban Chakraborty, Nov 10, 2015
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    • "Rps19-deficient zebrafish were generated using a morpholino, as previously described (Danilova et al., 2008; Torihara et al., 2011; Uechi et al., 2008). We also used mutant rpl11 hi3820bTg (Amsterdam et al., 2004), which has been characterized previously as a model of DBA in our lab (Danilova et al., 2011). "
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    ABSTRACT: Ribosomal biogenesis involves processing of pre-rRNA in assembly with ribosomal proteins (RPs). Deficiency of some RPs impairs processing and causes Diamond Blackfan Anemia (DBA) associated with anemia, congenital malformations, and cancer. p53 mediates many features of DBA but the mechanism of p53 activation remains unclear. Another hallmark of DBA is upregulation of adenosine deaminase (ADA) suggesting changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis consistent with the need to break and replace the faulty rRNA. We also found upregulation of dNTP synthesis, a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM/CHK1/2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion, and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements may be beneficial in DBA.
    Disease Models and Mechanisms 05/2014; 7(7). DOI:10.1242/dmm.015495 · 4.97 Impact Factor
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    • "Since the first report of extraribosomal functions of RPS1 in E. coli, additional extraribosomal functions of various RPs have been revealed by knockdown or knockout of RPs in various animal models. Knockdown of RPS19 in zebrafish leads to anemia [41, 42], and even more strikingly, knockdown of RPL11 in zebrafish results in developmental defects in the head [43]. Mutations in RPS19 or RPS20 in mice result in abnormal melanocyte proliferation and red blood cell hypoplasia [44]. "
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    ABSTRACT: Ribosomal proteins (RPs) have gained much attention for their extraribosomal functions particularly with respect to p53 regulation. To date, about fourteen RPs have shown to bind to MDM2 and regulate p53. Upon binding to MDM2, the RPs suppress MDM2 E3 ubiquitin ligase activity resulting in the stabilization and activation of p53. Of the RPs that bind to MDM2, RPL5 and RPL11 are the most studied and RPL11 appears to have the most significant role in p53 regulation. Considering that more than 17% of RP species have been shown to interact with MDM2, one of the questions remains unresolved is why so many RPs bind MDM2 and modulate p53. Genes encoding RPs are widely dispersed on different chromosomes in both mice and humans. As components of ribosome, RP expression is tightly regulated to meet the appropriate stoichiometric ratio between RPs and rRNAs. Once genomic instability (e.g. aneuploidy) occurs, transcriptional and translational changes due to change of DNA copy number can result in an imbalance in the expression of RPs including those that bind to MDM2. Such an imbalance in RP expression could lead to failure to assemble functional ribosomes resulting in ribosomal stress. We propose that RPs have evolved ability to regulate MDM2 in response to genomic instability as an additional layer of p53 regulation. Full understanding of the biological roles of RPs could potentially establish RPs as a novel class of therapeutic targets in human diseases such as cancer.
    Oncotarget 02/2014; 5(4). DOI:10.18632/oncotarget.1784 · 6.36 Impact Factor
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    • "By demonstrating protein haploinsufficiency, ribosome assembly defects and definitive erythroid differentiation defects, we have shown that our gene trap mutant mouse embryonic stem cell models faithfully recapitulate the major features of DBA. In our cellular model of DBA, both the Rps19 and Rpl5 mutants exhibit a severe defect in primitive erythropoiesis, which is in accord with others' findings in zebrafish and induced pluripotent stem cells [34]–[36]. We have expanded this finding to directly compare and contrast primitive and definitive erythropoiesis in quantifiable assays, which is a limitation in many other disease models. "
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    ABSTRACT: Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome caused by ribosomal protein haploinsufficiency. DBA exhibits marked phenotypic variability, commonly presenting with erythroid hypoplasia, less consistently with non-erythroid features. The p53 pathway, activated by abortive ribosome assembly, is hypothesized to contribute to the erythroid failure of DBA. We studied murine embryonic stem (ES) cell lines harboring a gene trap mutation in a ribosomal protein gene, either Rps19 or Rpl5. Both mutants exhibited ribosomal protein haploinsufficiency and polysome defects. Rps19 mutant ES cells showed significant increase in p53 protein expression, however, there was no similar increase in the Rpl5 mutant cells. Embryoid body formation was diminished in both mutants but nonspecifically rescued by knockdown of p53. When embryoid bodies were further differentiated to primitive erythroid colonies, both mutants exhibited a marked reduction in colony formation, which was again nonspecifically rescued by p53 inhibition. Cell cycle analyses were normal in Rps19 mutant ES cells, but there was a significant delay in the G2/M phase in the Rpl5 mutant cells, which was unaffected by p53 knockdown. Concordantly, Rpl5 mutant ES cells had a more pronounced growth defect in liquid culture compared to the Rps19 mutant cells. We conclude that the defects in our RPS19 and RPL5 haploinsufficient mouse ES cells are not adequately explained by p53 stabilization, as p53 knockdown appears to increase the growth and differentiation potential of both parental and mutant cells. Our studies demonstrate that gene trap mouse ES cells are useful tools to study the pathogenesis of DBA.
    PLoS ONE 02/2014; 9(2):e89098. DOI:10.1371/journal.pone.0089098 · 3.23 Impact Factor
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