Cells depleted for RPS19, a protein associated with Diamond Blackfan Anemia, show defects in 18S ribosomal RNA synthesis and small ribosomal subunit production

Abstract

The gene encoding the small subunit ribosomal protein 19 (RPS19) is mutated in about 25% of cases of the bone marrow failure syndrome Diamond Blackfan Anemia (DBA), a childhood disease characterized by failure of red cell production. In these cases DBA is inherited as an autosomal dominant trait and RPS19 haploinsufficiency is thought to cause the disease. To study the molecular pathogenesis of DBA we used siRNA to decrease the level of RPS19 in two human cell lines, HeLa cells and U-2 OS osteosarcoma cells. Cells with reduced RPS19 levels showed a dramatic reduction in the amounts of small 40S ribosome subunits and mature 80S ribosomes and an excess of large 60S subunits. These cells were defective in 18S rRNA production and accumulated 21S and 20S nuclear pre-rRNA molecules, suggesting that RPS19 is required for specific steps in rRNA processing. RPS19 depletion produced a reduction in steady-state levels of RPS6 and RPS16 via a post-transcriptional mechanism while the levels of RPL7 and RPL26 were unaltered, indicating that levels of ribosomal proteins are determined by subunit assembly. This has interesting implications for the pathogenesis of DBA suggesting that deficiency of any of the RPS proteins might have a similar effect and thus may be responsible for causing DBA. Finally in cell lines from DBA patients with mutations we find increased levels of 21S rRNA precursors but no abnormality in the ribosome profile on sucrose gradients or in the steady-state levels of RPS19 suggesting that some cells can partially compensate for the loss of one allele of RPS19. We conclude that defects in ribosome biogenesis may underlie the pathology of Diamond Blackfan Anemia.

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.