Figure 1.
Depletion of WBSCR22 reduces cell growth.
(A) Protein expression of siWBSCR22 and siNeg. transfected cells was determined by western blot analysis using anti-WBSCR22 and anti-tubulin antibodies. Proteins from 105 cells are loaded on each lane. (B) HeLa cells were transfected with siWBSCR22 or a control, siNeg, and the cell growth was monitored for five days. Average of three independent transfection experiments is shown.
Figure 2.
Polysome analysis of HeLa cells after depletion of WBSCR22.
(A) HeLa cells were transfected with siWBSCR22 and siNeg, the cytoplasmic cell extracts were prepared 72 h after transfection and centrifuged on 10-50% sucrose gradient. Absorbance at 254 nm was measured across the gradient, and the positions corresponding to the 40S, 60S and 80S ribosomal particles are indicated. (B) The ratio of 18S/28S rRNA from HeLa and HEK293 cells transfected with siRNAs. RNA was purified from cytoplasmic extracts of siWBSCR22 and siNeg cells, separated by electrophoresis and intensities of 18S and 28S rRNA were quantified. The P value is 0.04 using Student’s t-test. (C) Protein expression of siWBSCR22 and siNeg. transfected cells was determined by western blot analysis using anti-WBSCR22 and anti-tubulin antibodies.
Figure 3.
Analysis of pre-rRNA processing in WBSCR22-depleted cells.
(A) Pre-rRNA processing in HeLa cells according to Carron et al. [19]. (B) Northern blot analysis of HeLa (lanes 1-6) and HEK293 (lanes 7, 8) cells transfected with siWBSCR22 or siNeg. with the 5’-ITS1 probe. RNA was extracted 72 h after transfection and 3 µg of total (T), 6 µg of cytoplasmic (C) and 3 µg of nuclear (N) RNA were loaded on each lane. Lanes 7 and 8 show nuclear RNA. The positions of precursor rRNAs are indicated. (C) The amounts of pre-rRNAs in the nuclear fractions of HeLa and HEK293 cells were quantified by PhosphorImager and are represented relative to siNeg of appropriate cell line which was set as 1. Results shown are representative of three independent transfection experiments with standard deviations. The P value is 0.2 for 21S and 0.02 for 18S-E using Student’s t-test. (D) Northern blot analysis of rRNA from T (total), C (cytoplasmic) and N (nuclear) fractions with the 5’-ITS1 probe from HeLa cells transfected with siWBSCR22 and plasmids expressing E2Tag-WBSCR22 or epitope tag alone. Band corresponding to 18S-E pre-rRNA is shown. (E) Protein expression of siWBSCR22-depleted cells transfected with expression plasmid for E2Tag-WBSCR22 was determined by western blotting using anti-WBSCR22 and anti-tubulin antibodies.
Figure 4.
Complementation of yeast bud23Δ strain with human WBSCR22.
(A) Growth dilution assays of bud23Δ carrying plasmids pRS315, pRS315-BUD23 and pRS315-WBSCR22. Cultures were grown overnight and diluted to final optical density at OD600 0.1, from which further 10-fold serial dilutions were spotted. Plates were incubated at 30°C for 3 days. (B) Polysome profiles of bud23Δ carrying plasmids pRS315, pRS315-BUD23 and pRS315-WBSCR22. Cell lysates were centrifuged on 10-45% sucrose gradient. Absorbance at 254 nm was measured across the gradient, and the positions corresponding to the 40S, 60S and 80S ribosomal particles are indicated.
Figure 5.
Localization of the WBSCR22 protein within the cell.
(A) Subcellular localization of WBSCR22 by immunofluorescence analysis. The localization of endogenous protein in HeLa cells was analyzed by anti-WBSCR22 antibody. DAPI was used to stain the nucleus of the cell. (B) Total extracts of HeLa cells were centrifuged on 10-50% sucrose gradient at 27 000 rpm for 4 hours and fractionated. The 18S and 28S rRNAs were detected by ethidium bromide and the WBSCR22 protein was analyzed by western blotting using anti-WBSCR22 antibodies. (C) HeLa cell extracts were centrifuged on 10-50% sucrose gradient at 27 000 rpm for 13 hours and analyzed by western blotting.
Figure 6.
The WBSCR22 protein in WBS patient lymhoblastoid cell lines.
(A) Extracts from wild-type (E001 and E004) and three WBS patient-derived lymphoblastoid cell lines (GM13472, GM13473, GM13482) were immunoblotted to visualise the indicated proteins. (B) Quantification of western blot analysis. Average of three independent experiments with standard deviations is shown. The P value is in all cases below 0.005 using Student’s t-test.