ALBERT

Description: Diamond Blackfan anemia (DBA) is characterized by reduced proliferation and increased apoptosis of erythroid progenitors. Approximately 25% of patients are heterozygous for mutations in the gene encoding ribosomal protein S19 (RPS19). The molecular mechanisms behind DBA remain unclear and RPS19 was recently shown to interact with PIM1 kinase. To elucidate the phenotypic and cellular effects of this interaction we generated mice strains with the genotypes Rps19 +/− Pim1 +/+; Rps19 +/+ Pim1 −/− and Rps19 +/− Pim1 −/−. We analyzed the mice for peripheral blood and bone marrow parameters as well as apoptotic markers in primary bone marrow cells. The double mutant mice (Rps19 +/− Pim1 −/−) show normal growth and life span with increased white and red blood cell counts when compared to wild type, Rps19 heterozygous, and Pim1 null mice, respectively. Analysis of proteins in bone marrow cells shows that the combination of Rps19 insufficiency and Pim1 deficiency is associated with increased levels of Stat5 as well as the anti-apoptotic markers Mcl-1, Bcl-2 and Bcl-XL. The pro-apoptotic markers Bak, Caspase-3 and p21 show decreased levels whereas p53 remains unchanged. These findings suggest a co-operative effect of Rps19 insufficiency and Pim1 deficiency on cell proliferation in murine hematopoiesis.

Description: Diamond Blackfan Anemia (DBA) is a rare congenic anemia characterized by reduced erythrocyte precursors. Mutations in the ribosomal protein S19 (RPS19) gene are found in about 20% of patients, although it is unclear how this relates to the pathophysiology of the disease. Two transcripts have been reported for human RPS19 in the NCBI database (BC000023 and BC018616), which only differ in the length of the 5′UTR. Both transcripts predict translation to the same protein product and we therefore speculate that the extended 5′UTR may be of importance. While the short (569bp) transcript is well studied, the long (873bp) transcript has not been investigated. We determined the size and tissue distribution as a basic characterization of the long RPS19 transcript. Furthermore, we examined proteins interacting with the different RPS19 transcript variants. Two methods were used determine the size of long RPS19 transcript. Ribonuclease protection assays and 5′-RACE indicate a transcript length consistent with the reported sequence with a 5′UTR of 372bp. Interestingly the 5′UTR predicted in the long transcript contains sequence for a previously reported promoter, suggesting a second novel promoter. Tissue distribution was determined using reverse transcriptase PCR on isolated RNA from a panel of tissues including heart, bone marrow, fetal liver, liver, brain, fetal brain, muscle, kidney, uterus, adrenal gland, lungs, placenta, prostate testes, ovary, thymus, colon, lymphocytes and from K562 and HeLa cell lines. Analysis indicated a ubiquitously expression of the long RPS19 transcript. In order to investigate interactions between the RPS19 transcripts and regulatory proteins, a UV cross-linking assay was performed using in vitro transcribed RNA representing both short and long transcripts. A protein of 55 kDa from nuclear fractions prepared from the erythroid cell lines K562, UT-7 and Cos-1 cells was found to bind to the long RPS19 transcript but not to the short RPS19 transcript. Our results provide evidence for an additional long, RPS19 transcript containing an extended 5′UTR that is ubiquitously transcribed and indicates the existence of a second promoter for RPS19. We further show that a nuclear protein, present in two different erythroid cell lines, interacts with the long RPS19 transcript indicating a function for the extended 5′UTR. This data suggests additional regulatory pathways involving RPS19 mRNA which may be of relevance to the understanding of the molecular mechanisms behind DBA.

Description: 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.

Description: Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Forty percent of the patients are blood transfusion–dependent. Recent reports show that the ribosomal protein S19 (RPS19) gene is mutated in 25% of all patients with DBA. We constructed oncoretroviral vectors containing theRPS19 gene to develop gene therapy for RPS19-deficient DBA. These vectors were used to introduce the RPS19 gene into CD34+ bone marrow (BM) cells from 4 patients with DBA withRPS19 gene mutations. Overexpression of theRPS19 transgene increased the number of erythroid colonies by almost 3-fold. High expression levels of the RPS19transgene improved erythroid colony-forming ability substantially whereas low expression levels had no effect. Overexpression of RPS19 had no detrimental effect on granulocyte-macrophage colony formation. Therefore, these findings suggest that gene therapy for RPS19-deficient patients with DBA using viral vectors that express the RPS19gene is feasible.