Cells depleted for RPS19, a protein associated with Diamond Blackfan Anemia, show defects in 18S ribosomal RNA synthesis and small ribosomal subunit production
Introduction
Diamond Blackfan Anemia (DBA, OMIM #105650) is an inherited form of bone marrow failure that mainly affects the red blood cell lineage. The disease is usually diagnosed early in childhood. As well as failure of red cell development the disorder is associated with a variety of congenital abnormalities and a predisposition to malignancy (for review see Refs. [1], [2]). About 25–50% of DBA cases are familial [2], [3] while 25% of cases, whether familial or apparently sporadic, are associated with mutations in the gene RPS19 encoding small ribosomal protein RPS19 [4], [5]. Inheritance in the familial cases is usually autosomal dominant and in the case of the RPS19 families the pathogenesis appears to be due to haploinsufficiency [6]. Recently mutations in a second ribosomal protein encoding gene, RPS24, have been found to account for about 2% of RPS19 negative DBA cases [7], suggesting a common pathway in DBA pathogenesis.
The mechanism of pathogenesis of DBA is unknown. Mice heterozygous for an Rps19 gene deletion develop normally with no abnormalities in red cell development [8], [9]. Cell culture studies show that the defect is intrinsic to the red cell progenitors which fail to complete differentiation in response to erythropoietin [10]. Depleting the amount of RPS19 in CD34+ primitive hematopoietic progenitors caused a reduction in the proliferation of immature red cells [11], [12]. Since the only known genetic cause of DBA is mutation in small ribosomal protein genes, possible molecular mechanisms are defects in the synthesis or function of ribosomes. Interestingly, genes encoding proteins involved in ribosome synthesis are mutated in other inherited bone marrow failure syndromes [13]. Recently it has been shown in yeast that depletion of RPS19 severely affects the production of 40S ribosomal subunits and moreover that mutations in yeast RPS19 analogous to those causing DBA in humans lead to defective processing and accumulation of 18S ribosomal RNA precursors [14], [15].
Our hypothesis is that RPS19 haploinsufficiency causes DBA by disrupting pre rRNA processing and that certain cells, including red cell precursors are particularly sensitive to this disruption, either because they require high levels of ribosome production or because other cells can compensate for the loss of one RPS19 allele. Here we begin to test the first part of this hypothesis by studying the effect of RPS19 depletion on ribosome production. We use an siRNA approach to knockdown RPS19 production in HeLa cells. We find that decreasing the rate of production of RPS19 prevents the production of 18S rRNA and causes the accumulation of novel 21S and 20S precursors. Cells depleted for RPS19 have reduced numbers of 40S ribosome subunits and a dramatic reduction in the numbers of mature 80S ribosomes. Examination of rRNA processing and ribosome production in cell lines derived from lymphocytes from DBA patients showed some abnormalities in rRNA synthesis but normal levels of ribosomes.
Section snippets
Cell culture
HeLa cells were cultured in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum, antibiotics (100 μg of penicillin and 50 μg of streptomycin sulfate/ml) and 2 mM glutamine. U-2 OS cells were obtained from the American Type Culture Collection (HTB-96) and cultured in McCoy's 5a medium with 10% fetal bovine serum, 1.5 mM l-glutamine, 2.2 g/l sodium bicarbonate, and antibiotics (100 μg of penicillin and 50 μg of streptomycin sulfate/ml). Lymphoblast cell lines (LCL) were cultured
Depletion of RPS19 causes a decrease in 40S subunits and an accumulation of 60S subunits
DBA appears to be caused by haploinsufficiency for RPS19. Knocking down RPS19 expression in cell lines should create a similar situation with 50% or less of the normal amount of RPS19. We tested 5 siRNAs for their efficiency in knocking down RPS19 levels in growing HeLa cells. HeLa cells were transfected with the siRNAs and protein extracted 48 or 72 h later. At this stage no gross morphological changes were apparent in the cells. Western blotting shows that siRNAs #2, #3, #4 and #5 caused a
Discussion
The pathophysiology of Diamond Blackfan Anemia is not understood in detail. RPS19 haploinsufficiency causes the disease in about 25% of cases [2], [3], [4], [5], [6]. In this paper we have examined the consequences of RPS19 depletion on ribosome biogenesis in HeLa cells and in the osteosarcoma cell line U-2 OS.
Reducing the expression of RPS19 in HeLa or U-2 OS cells caused a drastic reduction in the rate of 18S RNA production while production of 28S RNA was only slightly decreased. This was
Acknowledgments
This work was supported by NIH/NIDKK R21 grant DK075443-01 and The Daniella Maria Arturi Foundation. We would like to thank Susan Baserga for her advice and encouragement and Mary Jane Eichenseer for the antibody purification.
References (26)
Diamond Blackfan Anemia: new paradigms for a “not so pure” inherited red cell aplasia
Semin. Hematol.
(2006)- et al.
Mutations in ribosomal protein S19 gene and Diamond Blackfan Anemia: wide variations in phenotypic expression
Blood
(1999) - et al.
Ribosomal protein S24 gene is mutated in Diamond-Blackfan Anemia
Am. J. Hum. Genet.
(2006) - et al.
Erythropoiesis in the Rps19 disrupted mouse: analysis of erythropoietin response and biochemical markers for Diamond-Blackfan anemia
Blood Cells Mol. Dis.
(2006) - et al.
Two-phase culture in Diamond Blackfan Anemia: localization of erythroid defect
Blood
(2005) - et al.
An RNA interference model of RPS19 deficiency in Diamond-Blackfan anemia recapitulates defective hematopoiesis and rescue by dexamethasone: identification of dexamethasone-responsive genes by microarray
Blood
(2005) - et al.
Deficiency of ribosomal protein S19 in CD34+ cells generated by siRNA blocks erythroid development and mimics defects seen in Diamond-Blackfan anemia
Blood
(2005) - et al.
Ribosomes and marrow failure: coincidental association or molecular paradigm?
Blood
(2006) - et al.
Specific role for yeast homologs of the Diamond Blackfan Anemia associated Rps19 protein in ribosome synthesis
J. Biol. Chem.
(2005) - et al.
Roles of eukaryotic ribosomal proteins in maturation and transport of pre-18S rRNA and ribosome function
Mol. Cell
(2005)
Expression of a novel regenerating gene product, Reg IV, by high density fermentation in Pichia pastoris: production, purification, and characterization
Protein Expr. Purif.
Diamond-Blackfan anemia: report of seven further mutations in the RPS19 gene and evidence of mutation heterogeneity in the Italian population
Blood Cells Mol. Dis.
Recent insights into the pathogenesis of Diamond-Blackfan Anaemia
Br. J. Haematol
Cited by (95)
Multi-transcriptome analysis following an acute skeletal muscle growth stimulus yields tools for discerning global and MYC regulatory networks
2022, Journal of Biological ChemistryMissense mutation in RPS7 causes Diamond-Blackfan anemia via alteration of erythrocyte metabolism, protein translation and induction of ribosomal stress
2022, Blood Cells, Molecules, and DiseasesRibosomal RNA Processing
2022, Encyclopedia of Cell Biology: Volume 1-6, Second EditionNuclear rRNA transcript processing versus internal transcribed spacer secondary structure
2015, Trends in Genetics