Analysis on the Correlation of RPS19 Expression Level to Diamond-Blackfan Anemia; Insights from the Expression Level of RPS19 Point Mutants in Erythroid Cell Lines.

Mutations in ribosomal protein S19 (RPS19) gene is closely related to Diamond-Blackfan anemia (DBA). We have found point mutation, deletion and insertion in RPS19 gene in DBA patients (Blood, 100: 2724–31, 2002). In our analysis of DBA patients, gene expression of RPS19 from these mutated genomes were down regulated. Retroviral transduction of RPS19 gene to patient bone marrow cells restored the ability to differentiate into erythroid in vitro (Mol Ther., 7: 613–22, 2003). However, the mechanisms of how these mutations in RPS19 associate with anemia remains unknown.

To analyze the mutated genome, we have made constructs of mutated RPS19 gene-expression vectors. From reported 56 different RPS19 mutations, we focused on the missense mutants and tried to characterize them. Flag-tagged twelve missense mutants (V15F, L18R, P47L, W52R, R56Q, S59F, A61Q, R62W, R101H, G120R, G127Q, and G131R) were exogenously expressed in several cell lines by retroviral vectors and were analysed by western blotting or FACS. When these 12 mutants were expressed in erythro-leukemic cell lines K562 and HEL, almost all the mutant proteins (except for G120R) were expressed at significantly low level compared to that of wild type. However, some of these mutants were expressed in human kidney epithelial cell line, 293T cells. These findings suggest that the expression level of RPS19 protein derived from mutated RPS19 genome is dependent on cell types, whether erythroid-lineage cells or others.

In order to analyze the function of RPS19, doxycycline (Dox)-inducible expression of siRNA against RPS19 was induced in K562 cells and observed growth arrest at day 4 after induction of siRNA by Dox. Expression level of RPS19 decreased to one tenth of the normal level. Cell cycle analysis and quantitative-PCR analysis revealed that the growth arrest of K562 cells expressing siRNA against RPS19 was due to the induction of cell cycle arrest at G1 phase (G0/G1 54 % vs 36% ) coincide with the up-regulation of p21 and p57 mRNA level. From these results, we could possibly suggested that, in DBA patients, RPS19 protein expression is not stable and the decreased level of RPS19 protein prolongs G1 phase of cell cycle especially in the course of differentiation on erythroid cells, which may contribute to the retarded production of red blood cells.

Our findings allow to understand the entire feature of DBA-associated RPS19 mutations especially on the erythorid differentiation and may help defining the therapeutic target to improve DBA.