Fig 1: PDCD2-P28S and -R34P variants show reduced protein levels and reduced PDCD2-uS5 complex formation. (A) Immunoblot analysis of total extracts prepared from HeLa cells transfected with DNA constructs expressing reference sequence PDCD2 (WT, lanes 1 to 3) or one of two patient variants, p.(Pro28Ser) (P28S, lanes 4 to 6) or p.(Arg34Pro) (R34P, lanes 7 to 9). HeLa cells were also co-transfected with a DNA construct expressing PABPN1-Flag to control for transfection efficiency. Anti-actin B (ActB) was used to normalize for protein loading (Lower). (B) Quantification of relative GFP-PDCD2 levels as determined by immunoblotting, showing reduced levels of patient variants. GFP-PDCD2/PABPN1-Flag ratios were normalized to actin B and expressed relative to wild-type PDCD2, which was set to 1.0. The data and error bars represent the average and SD from three independent experiments. ***P ≤ 0.001, as determined by Student’s t test. (C) Immunoblot analysis of total extracts (lanes 1 to 4) and anti-GFP precipitates (lanes 5 to 8) prepared from HeLa cells that were transiently transfected with the indicated versions of PDCD2 (lanes 2 to 4 and 6 to 8) or with a GFP control plasmid (lanes 1 and 5). (D) Quantification of uS5 levels recovered in GFP immunoprecipitates were normalized to the levels of reference GFP-PDCD2. Values were expressed relative to the reference (WT) version of PDCD2, which was set to 1.0. The data and error bars represent the average and SD from five independent experiments. *P ≤ 0.05, **P ≤ 0.01, as determined by Student’s t test.
Fig 2: Ribosome biogenesis in fetal fibroblasts from F2.1 critically depends on PDCD2 levels. (A) Two alternative pre-rRNA processing pathways in human cells. Mature 18S, 5.8S, and 28S rRNAs are produced from a single RNA polymerase I transcript (47S). Pathway 1 is initiated by the concomitant cleavage of the 45S pre-rRNA at sites A0 and 1, producing 43S and 41S pre-rRNAs, respectively. In contrast, pathway 2 starts in the ITS by cleavage at site 2, producing 30S and 32S pre-rRNAs. The 41S (pathway 1) and 30S (pathway 2) pre-rRNAs will ultimately be cleaved to produce the 21S precursor, which is further trimmed at the 3′ end to generate the 18S-E. 40S precursors containing the 18S-E pre-rRNA are exported to the cytoplasm where an endonucleolytic cleavage produces the mature 18S rRNA. The position of the probe ITS1, used to analyze the different pre-rRNA precursors, is shown on the 47S primary rRNA transcript. (B) Immunoblot analysis showing depletion of PDCD2 from primary fibroblasts of fetal control (lanes 1 and 2) and F2.1 (lanes 3 and 4), which were previously transfected with nontarget control (lanes 1 and 3) and PDCD2-specific (lanes 2 and 4) siRNAs. (C) Northern blot analysis of mature and precursor rRNAs using total RNA prepared from primary fibroblasts of control (lanes 1 and 2) and F2.1 (lanes 3 and 4) fetuses, which were previously transfected with nontarget (lanes 1 and 3) and PDCD2-specific (lanes 2 and 4) siRNAs. Pre-rRNAs species are indicated on the Right and were detected by using a probe complementary to sequences in the ITS1 region (A). (D and E) Levels of the 18S-E (D) and 21S (E) pre-rRNAs were normalized to the mature 28S rRNA and are expressed relative to values for fibroblasts of control fetus (Fet. Ctrl) treated with the nontarget control siRNA. Data and error bars represent the means and SD, respectively, from five independent experiments. *P ≤ 0.05, ****P ≤ 0.0001, as determined by Student’s t test.
Fig 3: Pedigrees for families with fetal hydrops and biallelic PDCD2 variants and analysis of reduced PDCD2 protein levels and reduced PDCD2 copurification with uS5 from F2.1 skin fibroblasts. (A) Pedigrees for Families 1 and 2 with identified PDCD2 variants indicated below each symbol. (B) Immunoblot of whole-cell lysates from F2.1 fetal skin fibroblasts and controls. Blotting was performed with antibodies to PDCD2 (Upper), uS5 (Middle), and GAPDH as loading control (Lower). (C) Quantification from immunoblotting demonstrating lower ratio of endogenous PDCD2 protein levels to GAPDH loading controls in F2.1 fibroblasts compared to controls. (D) Endogenous uS5 was immunoprecipitated from whole-cell lysates of F2.1 skin fibroblasts and controls using magnetic beads coupled with anti-uS5 antibody. Rabbit IgG was used as an isotype control. Anti-uS5 antibody (Upper) and anti-PDCD2 antibody (Middle) were used for target detection in the immunoprecipitates (IP) and the whole-cell lysates (Input). The prominent band just below 55 kDa corresponds to the heavy chain of the immunoprecipitation antibody. Anti-GAPDH antibody was used as loading control (Input, Lower). (E) Quantification from immunoblotting demonstrating lower ratio of endogenous PDCD2 co-immunoprecipitated with uS5 in F2.1 fibroblasts compared to controls. For C and E, the mean ± SD and single data points of three independent experiments are shown. One-way ANOVA with Dunnett’s correction was used for statistical analysis: *P ≤ 0.05; **P ≤ 0.01. Ctrl: Control, fet.: fetal.
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