Hematopoietic Mechanism in Diamond-Blackfan Anemia: Late Erythroid Development Is Not Affected by Ribosomal Protein S19 Deficiency.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 719-719
Author(s):  
Johan Flygare ◽  
Thomas Kiefer ◽  
Koichi Miyake ◽  
Taiju Utsugisawa ◽  
Isao Hamaguchi ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Liquid Culture ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Protein Levels ◽  
Diamond Blackfan Anemia ◽  
Cd34 Cells ◽  
Ribosomal Protein S19 ◽  
Erythroid Development ◽  
In Cells

Abstract Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. It is unknown how the ribosomal protein deficiency leads to anemia. We previously developed three lentiviral vectors expressing siRNA against RPS19 and one scramble control vector. All vectors also express GFP. We have previously shown that transduction of CD34+ bone marrow (BM) cells with the siRNA vectors reduced RPS19 mRNA levels, resulting in formation of fewer erythroid colonies. In the present study, we have demonstrated downregulation of RPS19 protein in siRNA treated cells. RPS19 protein levels decreased over time and were reduced to 40-60% of normal in cells transduced with all three siRNA vectors 5 days after transduction. We asked which stage of erythroid development is most affected by RPS19 deficiency. After 7 days in liquid culture supporting erythroid differentiation Glycophorin A (GlyA) and CD71 expression was examined by FACS. RPS19-silenced as well as DBA patient CD34+ cells exhibited a block in erythroid differentiation seen as an increased fraction of GlyAneg CD71low cells while the fractions of CD71high GlyAintermediate and GlyAhigh cells decreased. We cultured untransduced CD34+ cells in liquid culture for 7 days and isolated CD71high GlyA intermediate cells that are highly enriched in CFU-E and do not contain BFU-E. These cells were transduced with RPS19 siRNA vectors. Further erythroid maturation from CFU-E (CD71high GlyAintermediate) to more mature erythroid cells (GlyAhigh) was not affected by RPS19 silencing suggesting that the main block in erythroid development occurs prior to the CFU-E formation. The failure of erythroid differentiation correlated with the decrease in RPS19 protein levels. Transduction with a lentivirus expressing an siRNA-resistant RPS19 transcript rescued both the erythroid progenitor proliferation and differentiation, showing that the DBA-like phenotype is specific to the RPS19 deficiency. Finally we cultured the cells in liquid medium supporting both erythroid and myeloid differentiation. Proliferation decreased while the ratio of mature myeloid to erythroid cells increased 3 fold in cells transduced with the 2 most efficient siRNA-vectors, meaning that erythroid development is more sensitive to low RPS19 levels than myeloid development. When RPS19 is downregulated to intermediate levels, erythroid differentiation and proliferation of erythroid progenitors is severely reduced. More severe reduction of RPS19 impairs proliferation of myeloid progenitors as well, leading to a reduced output of myeloid progeny. Although our data cannot rule out hypothetical extraribosomal mechanisms we suggest that the major clinical findings in RPS19 deficient DBA can be explained by insufficient protein translation. This study shows for the first time that RPS19 protein downregulation decreases the proliferative capacity of hematopoietic progenitors leading to an early defect in erythroid development.

P53 is Activated without RPL11 Upregulation in Diamond-Blackfan Anemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3432-3432
Author(s):  
Hong-Yan Du ◽  
M. Tarek Elghetany ◽  
Blanche P Alter ◽  
Akiko Shimamura
Keyword(s):  
Bone Marrow ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Mrna Levels ◽  
Protein Levels ◽  
Diamond Blackfan Anemia ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
P53 Activation ◽  
Ribosomal Stress

Abstract Abstract 3432 Diamond-Blackfan anemia (DBA) is an autosomal dominantly inherited bone marrow failure syndrome characterized by red cell aplasia, physical anomalies, and cancer predisposition. DBA is caused by mutations resulting in haploinsufficiency of genes encoding ribosomal proteins. p53 is activated in the erythroid lineage following reduction of ribosomal protein expression; however the mechanism whereby ribosomal stress results in p53 activation in DBA remains unclear. RPL11 has been proposed to play a central role in p53 activation following ribosomal stress. Reduced expression of individual small ribosomal subunit proteins in a tumor cell line resulted in increased translation of RPL11. Excess free RPL11 can bind and inactivate HDM2, an E3 ubiquitin ligase targeting p53 for degradation. The recent demonstration that cellular responses to ribosomal perturbations vary widely between different tissues raised the question of whether RPL11 upregulation contributes to p53 activation following ribosomal stress in hematopoietic progenitors. To address this question, we modeled DBA in human CD34+ cells. Since RPS19 is the most commonly mutated gene in DBA, we used lentiviral vectors expressing short hairpin RNAs to knock down RPS19 expression in primary human CD34+ cells. RPS19 protein levels were reduced to about 50% of control levels in a manner reflecting the haploinsufficient state in DBA. RPS19 depletion resulted in elevated p53 protein levels and increased mRNA levels of p21, a transcriptional target of p53. Total p53 mRNA levels and p53 mRNA translational activity remained unchanged consistent with a post-transcriptional mechanism for p53 activation. Although total RPL11 mRNA levels were not diminished following RPS19 depletion, RPL11 protein levels were significantly decreased consistent with post-transcriptional downregulation. Depletion of RPS19 in human CD34+ cells did not affect polysome loading of RPL11 mRNA. Reduction of additional ribosomal proteins also accompanied RPS19 knockdown consistent with coordinate regulation of multiple ribosomal protein levels. Corticosteroids, which improve anemia in the majority of DBA patients, did not prevent p53 activation, nor did this improve RPS19 or RPL11 protein levels. Expression of p53 was also assessed in bone marrow biopsy slides from 26 DBA patients with the following genotypes: RPS19 (18), RPS24 (2), RPS26 (2), RPS10 (1), RPS17 (1), RPS7 (1), and RPL11 (1). p53 was over-expressed in all but one patient (RPS26), and was clearly over-expressed in the DBA patient harboring the RPL11 mutation. In summary, we find that p53 activation in DBA does not require upregulation of RPL11 translation or elevated RPL11 protein levels. p53 activation persists in DBA caused by RPL11 deficiency. Corticosteroids do not improve ribosomal protein levels nor do they prevent p53 activation. Disclosures: No relevant conflicts of interest to declare.

The First Homozygous RPS19 Mutation Identified in a Diamond-Blackfan Anemia Patient Is Not Deleterious.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3548-3548
Author(s):  
Aurore Cretien ◽  
Patricia Rince ◽  
Thierry Leblanc ◽  
Rolande Ducrocq ◽  
Alexis Proust ◽  
...  
Keyword(s):  
Protein Expression ◽  
Ivory Coast ◽  
Iron Chelation ◽  
Erythroid Differentiation ◽  
Homozygous Mutation ◽  
Erythroid Cells ◽  
Cloning Efficiency ◽  
Erythroid Progenitors ◽  
Diamond Blackfan Anemia ◽  
Cd34 Cells

Abstract Mutations in ribosomal protein S19 (RPS19) gene have been found in 25% of patients affected with Diamond-Blackfan anemia (DBA). Two of the RPS19 mutations identified in DBA patients are located in the sequence upstream of the translational start site: a missense mutation g->t at −460 and a 4 bp insertion, gcca, 3 nt upstream of the 5′ end of the RPS19 promoter. All of the RPS19 mutations identified to date are expressed in the heterozygous state. In the present study, in a DBA patient from Ivory Coast we identified the gcca insertional mutation on both alleles. Diagnosis of DBA was made at the age of 3 months. No malformations were noted. The patient responded neither to steroid nor to IL-3 therapies. The patient was regularly transfused but without an effective iron chelation therapy. As a consequence DBA was complicated by a severe hemochromatosis with thyroid, parathyroid, and liver damage. The patient was enrolled into a metoclopramide protocol in our hospital when he was 12 year old. He had a partial response, with increased time intervals between transfusions. While analysis of the RPS19 gene did not identify a mutation in the coding sequence, the 4 bp insertion, gcca at nt − 631 was noted on both alleles. Mother was heterozygous for this mutation and strikingly the father was homozygous. Both parents are apparently healthy. In screening of 200 Caucasian control chromosomes and 100 chromosomes from Ivory Coast we did not identify the 4bp insertion, ruling out the possibility that it is a common polymorphism. A parental disomy was eliminated by a genescan microsatellite analysis using the microsatellites: D19S200, D19S197, and LIPE. To evaluate the effect of this mutation on erythroid differentiation, we isolated 30,000 CD34+ cells from peripheral blood of the patient and normal individuals. CD34+ cells were cultured for 7 days in methylcellulose with EPO, SCF, and IL-3. At day 7, erythroid colonies were isolated and cultured for additional 3 and 5 days in liquid medium. At D7, D10 and D12, aliquots of cells were collected and cloning efficiency and apoptosis was quantitated. RPS19 mRNA was assayed by quantitative RT-PCR and level of protein expression determined by Western blot analysis. The cloning efficiency of DBA erythroid progenitors was decreased by 2.6 fold compared to normal between D7 and D10 and by 3.5 fold between D10 and D12. No difference in apoptosis was noted between DBA and normal erythroid progenitors. Strikingly, during terminal erythroid differentiation, RPS19 mRNA and protein expression levels was similar in the DBA and normal erythroid cells. The presence of the homozygous mutation in the healthy father in conjunction with normal expression of RPS19 in the DBA erythroid cells imply that the gcca insertion at nt −631 is not deleterious and cannot by itself account for the DBA phenotype of our patient.

Transcriptome Analysis of Erythroid Cells Cultured from Diamond Blackfan Anemia Patients with Ribosomal and GATA1 Mutations Reveals Dysregulation of Inflammatory Response Genes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3605-3605
Author(s):  
Kelly O'Brien ◽  
Adrianna Vlachos ◽  
Stacie M. Anderson ◽  
Crystiana Tsujiura ◽  
Lionel Blanc ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Ribosomal Protein ◽  
Blot Analysis ◽  
Full Length ◽  
Heme Biosynthesis ◽  
Biosynthesis Pathway ◽  
Erythroid Cells ◽  
Diamond Blackfan Anemia ◽  
Cd34 Cells ◽  
Gata1 Mutation

Abstract Diamond Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome characterized by red cell aplasia, usually without perturbation of other hematopoietic lineages. DBA patients are generally diagnosed during infancy or early childhood, have a high frequency of congenital anomalies, and a predisposition to cancer. Approximately 65% of DBA patients have identifiable heterozygous gene mutations or deletions in ribosomal protein genes. Additionally, mutations in GATA1, a key transcription factor in erythropoiesis, have been demonstrated in DBA patients (Sankaran VG et al. JCI. 122: 2439-43, 2012; Ludwig LS et al. Nat Med 20(7):748-53, 2014; Klar J et al. Br J Haem 166(6): 949-51, 2014). Despite our knowledge of the molecular pathology, the mechanism underlying the erythroid failure in DBA is not well understood, largely due to the inherent difficulties in studying primary erythroid cells from DBA patients. Furthermore, DBA patients with GATA1 mutations have not been well characterized and it remains unclear whether the pathogenic mechanisms of GATA1 DBA are similar to that of ribosomal DBA. We designed a two-step, 14 day in vitro culture system to generate erythroid cells from CD34+ stem/progenitor cells isolated from <10ml of peripheral blood (PB) collected from patients enrolled in the DBA Registry of North America. Patients with RPL5, RPS17(n=2), RPS24 and RPL35a, GATA1 mutations (n=2), and patients without identifiable mutations (n=3) were studied and compared to healthy control PB CD34+ cells. At day 14, we routinely obtain at least 10x fewer CD235+ erythroid cells (proerythroblasts and basophilic erythroblasts) in DBA cultures vs. controls (1x106 vs. 1x107 from 1x104 CD34+ cells). Further, a 2-7 day delay in the acquisition of CD235 was observed. Gene expression analyses was performed by Affymetrix GeneChip Human Gene ST Arrays and RNASeq to analyze protein coding and long non-coding RNA transcripts in cells from day 14 of erythroid cultures. Ingenuity pathway analysis (IPA) of the dysregulated genes between patients with ribosomal mutations, GATA1 mutations and controls revealed that many of the genes dysregulated in DBA were involved in multiple leukocyte migration and inflammatory signaling pathways, including the IL8, IL1R1, CXCR4, ICAM3, MPO, TNFSF10, and TLR4 genes with IL6, TNF, and lipopolysaccharide as top upstream regulators. Notably, the dysregulated genes in GATA1 patient cells largely overlapped that of the DBA patients with ribosomal mutations, including disruption of the leukocyte migration and inflammatory response genes. Patients with GATA1 and ribosomal protein mutations shared a number of dysregulated erythroid genes including AHSP, FAM132B, HEMGN, and TRIM10, however GATA1 patient cells showed GATA1 as the top upstream regulator and additionally showed dysregulation of heme biosynthesis pathway genes, including the ALAS2, FECH, CPOX, PPOX, and UROS. Few studies have looked at the pathogenesis of DBA in patients with GATA1 mutations. The DBA-associated splice donor mutation in GATA1 results in the exclusive expression of the short form of the GATA1 protein (GATA1s), which lacks the transactivation domain. Western blot analysis of control erythroid cells showed expression of both full length GATA1 and GATA1s, with the full length protein predominating. In cells from the patients with the GATA1 mutation, only GATA1s protein was expressed and the level of GATA1s exceeded the combined level of both GATA1 isoforms in control cells. Erythroid cells from a patient with an RPL5 mutation showed GATA1 protein levels comparable to controls, indicating the DBA phenotype of the RPL5 patient is not due to altered translation of full length GATA1. Northern blot analysis demonstrated that the GATA1 mutation did not affect ribosomal RNA processing. In summary, our transcriptome analyses have revealed novel insights into the molecular pathogenesis of ribosomal and GATA1-mutated DBA. Significant dysregulation of inflammatory gene pathways in DBA patients with both ribosomal protein and GATA1 mutations were observed, suggesting a shared pathological mechanism. Furthermore, the heme biosynthesis pathway was uniquely disrupted in patients with GATA1 mutations. Further investigating the inflammatory pathways in DBA may reveal novel targets for therapeutic development. Disclosures No relevant conflicts of interest to declare.

HSP70, the Key to Account for Erythroid Tropism of Diamond-Blackfan Anemia?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 671-671
Author(s):  
Marc Gastou ◽  
Sarah Rio ◽  
Mickael Dussiot ◽  
Narjesse Karboul ◽  
Thierry Leblanc ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Critical Role ◽  
Erythroid Differentiation ◽  
Expression Level ◽  
Hsp70 Expression ◽  
Erythroid Cells ◽  
Diamond Blackfan Anemia ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
P53 Activation

Abstract Diamond-Blackfan anemia (DBA) was the first ribosomopathy identified and is characterized by a moderate to severe, usually macrocytic aregenerative anemia associated with congenital malformations in 50% of the DBA cases. This congenital rare erythroblastopenia is due to a blockade in erythroid differentiation between the BFU-e and CFU-e stages. The link between a haploinsufficiency in a ribosomal protein (RP) gene that now encompass 15 different RP genes and the erythroid defect is still to be fully defined. Recently, mutations in TSR2 and GATA1 genes have been identified in a few DBA families. The GATA1 gene encodes for the major transcription factor critical for erythropoiesis and mutation in this gene that lead to loss of expression of the long form of the protein, necessary for the erythroid differentiation accounts for erythroblastopenia of DBA phenotype. Our group and others (Dutt et al., Blood 2011) have shown previously that p53 plays an important role in the DBA erythroblastopenia, inducing cell cycle arrest in G0/G1 and depending on the nature of RP gene mutation, a delayed erythroid differentiation and an increased apoptosis. Indeed, we identified two distinct DBA phenotypes (H. Moniz, M. Gastou, Cell Death Dis, 2012): a haploinsufficiency in RPL5 or RPL11 reduced dramatically the erythroid proliferation, delayed the erythroid differentiation, and markedly increased apoptosis, while RPS19 haploinsufficiency while reduced the extent of erythroid proliferation without inducing significant apoptosis. While p53 pathway has been found to be activated in RP haploinsufficient erythroid cells in DBA patients or shRNA-RPS19, -RPL5, or -RPL11 infected CD34+ erythroid cells, the intensity of the p53 activation pathway (p21, BAX, NOXA) is different depending on the mutated RP gene. Since the differences between the two phenotypes involved the degree of apoptosis we hypothesized that HSP70, a chaperone protein of GATA1 may play a key role in the erythroid defect of DBA. Indeed, HSP70 protects GATA1 from the cleavage by the caspase 3, a protease activated during erythroid differentiation and as such reduced levels of HSP70 related to a RP haploinsufficiency could account for increased apoptosis and delayed erythroid differentiation of erythroid cells in DBA. Indeed, a defect in RPL5 or RPL11 decreased dramatically the expression level of HSP70 and GATA1 in primary human erythroid cells from DBA patients and following in vitro knockdown of the proteins in CD34+ cells by RPL5 or RPL11 shRNA. Importantly, RPS19 haploinsufficiency did not exhibit this effect in conjunction with normal levels of HSP70 expression. Furthermore, we found that the decreased expression level of HSP70 was independent on the p53 activation. Strikingly, HSP70 was noted to be degraded by the proteasome since the bortezomib, the MG132, or the lactacystin were able to restore both the HSP70 expression level and intracellular localization in the cell. The lentiviral infection of haploinsufficient RPL5 or RPL11 cord blood CD34+ cells with a wild type HSP70 cDNA restored both the erythroid proliferation and differentiation confirming a critical role for HSP70 in the erythroid proliferation and differentiation defect in the RPL5 or RPL11 DBA phenotypes. The loss of HSP70 may explain the loss of GATA1 in DBA and also the erythroid tropism of the DBA disease. Restoration of the HSP70 expression level may be a viable and novel therapeutic option for management of this debilitating and difficult to manage erythroid disorder. Disclosures No relevant conflicts of interest to declare.

scholarly journals In Vitro Analysis of Erythroid Cells Derived from the Culture of Diamond Blackfan Anemia Patient CD34+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 744-744
Author(s):  
Kelly A O'Brien ◽  
Stacie M Anderson ◽  
Jason Farrar ◽  
Adrianna Vlachos ◽  
Eva Atsidaftos ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Blot Analysis ◽  
Target Genes ◽  
Erythroid Differentiation ◽  
Full Length ◽  
Erythroid Cells ◽  
Ribosomal Protein Genes ◽  
Diamond Blackfan Anemia ◽  
Gata1 Mutation

Abstract Diamond Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome characterized by severe macrocytic anemia, usually without perturbation of other hematopoietic lineages. DBA patients are generally diagnosed during infancy or early childhood, have a high frequency of congenital anomalies, and a predisposition to cancer. Approximately 65% of DBA patients have heterozygous mutations or deletions in ribosomal protein genes. Additionally, mutations in the GATA1 gene, which encodes the GATA1 erythroid transcription factor, have been demonstrated in two DBA patients (Sankaran VG et al. J Clin Invest. 122: 2439-43, 2012). The genetic cause of DBA in the remaining 35% of patients is unknown. Despite our knowledge of the genotypes, the mechanism underlying the erythroid failure in DBA is not completely understood. This is largely due to the inability to study primary erythroid cells from DBA patients. To begin to delineate the mechanisms regulating erythroid differentiation in DBA, we developed an in vitro culture system starting with CD34+ progenitor cells isolated from approximately 20 ml of DBA patient peripheral blood collected prior to transfusion at the DBA Registry of North America. Using this system, we have characterized DBA patients with mutations in large (RPL5) and small (RPS17) subunit ribosomal protein genes, one individual with a mutation in the GATA1 gene, and several patients with unknown mutations. At the end of the culture, we routinely obtained 6x107 CD235+ erythroid cells from an initial population of 5x104 control CD34+ progenitor cells. In contrast to control cells, cells from the DBA patients exhibited a significantly reduced growth rate and generated approximately 100-fold fewer CD235+ erythroid cells, with a two day delay in the acquisition of the CD235 marker. Using flow cytometry, we isolated populations of CD41-, CD44+, CD235+ erythroid cells from both control and patient cell cultures from which we extracted RNA. This allowed the first time comparison of mRNA expression in DBA erythroid cells. Protein coding and long non-coding RNA transcripts were compared using Affymetrix GeneChip Human Gene ST Arrays and RNASeq. Compared to controls, CD235+ cells from the patient with the RPL5 mutation showed decreased levels of the GAS5 (growth arrest) and NOP56 (large ribosomal subunit assembly) mRNAs, as well as small nucleolar RNAs. Ingenuity Pathway Analysis (IPA) identified the tRNA charging and RNA Polymerase II assembly pathways as significantly perturbed in this patient. The DBA-associated splice donor mutation in GATA1 results in the exclusive expression of the short form of the GATA1 protein (GATA1s), which lacks the transactivation domain. Western blot analysis of CD235+ control cells showed expression of both full length GATA1 and GATA1s, with the full length protein predominating. In the patient cells with the GATA1 mutation, only the GATA1s protein was expressed and exceeded the combined level of both GATA1 isoforms in the controls. Northern blot analysis demonstrated that the GATA1 mutation did not affect ribosomal RNA processing. The microarray and RNASeq expression profiles of the patient with the GATA1 mutation differed significantly from controls and from that of the patient with the RPL5 mutation. Many known GATA1 target genes including SLC4A1, AHSP, and TRIM10 were down regulated in the CD235+ erythroid cells of the patient with the GATA1mutation compared to control cells, clearly indicating that these genes depend on full length GATA1 for activation. IPA identified the heme biosynthesis pathway as significantly perturbed in this patient and GATA1 as the top regulator. In summary, we have shown that DBA patient cells show decreased proliferative and erythroid differentiation capabilities in vitro. RNA transcript analysis in DBA patient cells has revealed significant differences between DBA patients with a ribosomal protein gene mutation and a mutation in the GATA1 gene. These data delineate multiple signaling pathways or mechanisms involved in DBA and erythroid differentiation. Finally, we have demonstrated that many GATA1 target genes depend on full length GATA1 for activation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulation of globin-heme balance in Diamond-Blackfan anemia by HSP70/GATA1

Blood ◽  
2019 ◽  
Vol 133 (12) ◽  
pp. 1358-1370 ◽  
Author(s):  
Sarah Rio ◽  
Marc Gastou ◽  
Narjesse Karboul ◽  
Raphaёl Derman ◽  
Thunwarat Suriyun ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Diamond Blackfan Anemia ◽  
Heme Synthesis ◽  
Cd34 Cells ◽  
Free Heme ◽  
Globin Synthesis

AbstractDiamond-Blackfan anemia (DBA) is a congenital erythroblastopenia that is characterized by a blockade in erythroid differentiation related to impaired ribosome biogenesis. DBA phenotype and genotype are highly heterogeneous. We have previously identified 2 in vitro erythroid cell growth phenotypes for primary CD34+ cells from DBA patients and following short hairpin RNA knockdown of RPS19, RPL5, and RPL11 expression in normal human CD34+ cells. The haploinsufficient RPS19 in vitro phenotype is less severe than that of 2 other ribosomal protein (RP) mutant genes. We further documented that proteasomal degradation of HSP70, the chaperone of GATA1, is a major contributor to the defect in erythroid proliferation, delayed erythroid differentiation, increased apoptosis, and decreased globin expression, which are all features of the RPL5 or RPL11 DBA phenotype. In the present study, we explored the hypothesis that an imbalance between globin and heme synthesis may be involved in pure red cell aplasia of DBA. We identified disequilibrium between the globin chain and the heme synthesis in erythroid cells of DBA patients. This imbalance led to accumulation of excess free heme and increased reactive oxygen species production that was more pronounced in cells of the RPL5 or RPL11 phenotype. Strikingly, rescue experiments with wild-type HSP70 restored GATA1 expression levels, increased globin synthesis thereby reducing free heme excess and resulting in decreased apoptosis of DBA erythroid cells. These results demonstrate the involvement of heme in DBA pathophysiology and a major role of HSP70 in the control of balanced heme/globin synthesis.

Ribosomal protein S19 expression during erythroid differentiation

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 318-324 ◽  
Author(s):  
Lydie Da Costa ◽  
Goutham Narla ◽  
Thiébaut-Noel Willig ◽  
Luanne L. Peters ◽  
Marilyn Parra ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Regulatory Element ◽  
Erythroid Differentiation ◽  
Gene Encoding ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Diamond Blackfan Anemia ◽  
Sequence Elements ◽  
Ribosomal Protein S19 ◽  
Terminal Erythroid Differentiation

Abstract The gene encoding ribosomal protein S19 (RPS19) has been shown to be mutated in 25% of the patients affected by Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. As the role of RPS19 in erythropoiesis is still to be defined, we performed studies on RPS19 expression during terminal erythroid differentiation. Comparative analysis of the genomic sequences of human and mouse RPS19genes enabled the identification of 4 conserved sequence elements in the 5′ region. Characterization of transcriptional elements allowed the identification of the promoter in the human RPS19 gene and the localization of a strong regulatory element in the third conserved sequence element. By Northern blot and Western blot analyses of murine splenic erythroblasts infected with the anemia-inducing strain Friend virus (FAV cells), RPS19 mRNA and protein expression were shown to decrease during terminal erythroid differentiation. We anticipate that these findings will contribute to further development of our understanding of the contribution of RPS19 to erythropoiesis.

scholarly journals Human RPS19, the gene mutated in Diamond-Blackfan anemia, encodes a ribosomal protein required for the maturation of 40S ribosomal subunits

Blood ◽  
2006 ◽  
Vol 109 (3) ◽  
pp. 980-986 ◽  
Author(s):  
Johan Flygare ◽  
Anna Aspesi ◽  
Joshua C. Bailey ◽  
Koichi Miyake ◽  
Jacqueline M. Caffrey ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Diamond Blackfan Anemia ◽  
Cd34 Cells ◽  
Specific Step ◽  
Protein Functions ◽  
Ribosomal Protein S19 ◽  
Maturation Defect ◽  
First Year Of Life

Abstract Diamond-Blackfan anemia (DBA) typically presents with red blood cell aplasia that usually manifests in the first year of life. The only gene currently known to be mutated in DBA encodes ribosomal protein S19 (RPS19). Previous studies have shown that the yeast RPS19 protein is required for a specific step in the maturation of 40S ribosomal subunits. Our objective here was to determine whether the human RPS19 protein functions at a similar step in 40S subunit maturation. Studies where RPS19 expression is reduced by siRNA in the hematopoietic cell line, TF-1, show that human RPS19 is also required for a specific step in the maturation of 40S ribosomal subunits. This maturation defect can be monitored by studying rRNA-processing intermediates along the ribosome synthesis pathway. Analysis of these intermediates in CD34− cells from the bone marrow of patients with DBA harboring mutations in RPS19 revealed a pre-rRNA–processing defect similar to that observed in TF-1 cells where RPS19 expression was reduced. This defect was observed to a lesser extent in CD34+ cells from patients with DBA who have mutations in RPS19.

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