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

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.

Figure 1

Pre-rRNA processing in human cells. The major rRNA-processing pathways in human cells as initially derived from Hadjiolova et al and modified by Rouquette et al. Mature rRNA species are shown as filled boxes: 18S, ■; 5.8S, ▩; and 28S, ▨. External and internal transcribed sequences are shown as lines and are labeled above the primary transcript. Cleavage sites are designated with numbered and lettered arrows. Oligonucleotide probes used in Northern blot analysis are shown as lines below the primary transcript and are labeled with Greek letters. Two alternative pathways observed in human cells are shown below the 45S′ pre-rRNA that differ in the order of cleavages 1 and 2.

Figure 2

Northern blot analysis demonstrates abnormal pre-rRNA processing in TF-1 cells depleted of RPS19. Total RNA was isolated from TF-1 cells, fractionated on 1.5% formaldehyde-agarose gels, transferred to zetaprobe, and hybridized with oligonucleotides complementary to different regions of the rRNA primary transcript. The siRNAs present in each cell line are listed above each lane. Cell lines in lanes labeled A and B express 2 different siRNAs targeting RPS19. Sc indicates scrambled siRNA. Cell lines were grown for 4 days in the presence (+DOX) or absence (−DOX) of 0.5 μg/mL DOX. Panels are designated according to the oligonucleotide used for hybridization. Pre-rRNAs hybridizing with the different oligonucleotide probes are designated with arrows to the right or left of the panels. Illustrations of rRNA species hybridizing with different probes are included to the sides of each image. Filled boxes represent mature rRNAs: 18S, ■; 5.8S, ▩; 28S, ▨.

Figure 3

Pulse-chase analysis demonstrates abnormal pre-rRNA processing in TF-1 cells depleted of RPS19. Pulse-chase was carried out as described in “Materials and methods.” TF-1 cells infected with lentiviruses containing either siRNA B targeted to RPS19 (RPS19) or a scrambled siRNA (scrambled) were grown for 4 days in the presence (+) or absence (−) of DOX. Chase periods are shown above each lane.

Figure 4

Altered polysome profiles in TF-1 cells depleted of RPS19. Cells extracts were prepared for polysome analysis as described in “Materials and methods.” TF-1 cells infected with a lentivirus containing siRNA A targeted to RPS19 were grown for 4 days in the presence (+DOX) or absence (−DOX) of 0.5 μg/mL DOX. Extracts were layered on 15% to 55% sucrose gradients, and centrifugation was carried out for 5 hours at 67 000g. Gradients were fractionated using an ISCO-type 185 gradient fractionator, and absorbance at 254_nm was monitored with a UA-6 absorbance detector.

Figure 5

Northern blot analysis of CD34⁻ BM cells reveals abnormal pre-rRNA processing in patients with DBA who have mutations in RPS19. Total RNA was isolated from CD34⁻ cells and prepared for Northern blot analysis as described in Figure 2. Panels are designated according to oligonucleotides used for hybridization. Patients with DBA who have mutations in RPS19 are designated DBA-7⁻ and DBA-8⁻, while patients with normal RPS19 are designated DBA-1⁺ to DBA-5⁺. Samples from DBA patients labeled DBA-7⁻ and DBA-8⁻ have a chromosome breakpoint mutation in RPS19 and a complete deletion of RPS19, respectively. Ratios listed in Table 1 are derived from phosphorimage analysis of signals for the RNA species listed. Not all samples listed in Table 1 are shown in here.

Figure 6

Northern blot analysis displays defective pre-rRNA processing in CD34⁺ cells from patients with DBA who have mutations in RPS19. The figure shows a representative Northern blot using total RNA isolated from CD34⁺ cells and prepared for Northern blot analysis as described in Figure 2. Pre-rRNAs were hybridized with oligonucleotide γ (Figure 1) to examine the ratio of 21S to 18SE pre-rRNA.