Depletion of WBSCR22 reduces cell growth.

<p>(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.</p

The WBSCR22 protein in WBS patient lymhoblastoid cell lines.

<p>(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.</p

Complementation of yeast <i>bud23Δ</i> strain with human WBSCR22.

<p>(A) Growth dilution assays of <i>bud23Δ</i> carrying plasmids <i>pRS315</i>, <i>pRS315-BUD23</i> and <i>pRS315</i>-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 <i>bud23Δ</i> carrying plasmids <i>pRS315</i>, <i>pRS315-BUD23</i> and <i>pRS315</i>-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.</p

Polysome analysis of HeLa cells after depletion of WBSCR22.

<p>(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.</p

Analysis of pre-rRNA processing in WBSCR22-depleted cells.

<p>(A) Pre-rRNA processing in HeLa cells according to Carron et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075686#B19" target="_blank">19</a>]. (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.</p

Localization of the WBSCR22 protein within the cell.

<p>(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.</p

The Stability of Ribosome Biogenesis Factor WBSCR22 Is Regulated by Interaction with TRMT112 via Ubiquitin-Proteasome Pathway

<div><p>The human WBSCR22 protein is a 18S rRNA methyltransferase involved in pre-rRNA processing and ribosome 40S subunit biogenesis. Recent studies have shown that the protein function in ribosome synthesis is independent of its enzymatic activity. In this work, we have studied the WBSCR22 protein interaction partners by SILAC-coupled co-immunoprecipitation assay and identified TRMT112 as the interaction partner of WBSCR22. Knock-down of TRMT112 expression decreased the WBSCR22 protein level in mammalian cells, suggesting that the stability of WBSCR22 is regulated through the interaction with TRMT112. The localization of the TRMT112 protein is determined by WBSCR22, and the WBSCR22-TRMT112 complex is localized in the cell nucleus. We provide evidence that the interaction between WBSCR22/Bud23 and TRMT112/Trm112 is conserved between mammals and yeast, suggesting that the function of TRMT112 as a co-activator of methyltransferases is evolutionarily conserved. Finally, we show that the transiently expressed WBSCR22 protein is ubiquitinated and degraded through the proteasome pathway, revealing the tight control of the WBSCR22 protein level in the cells.</p></div

WBSCR22 interacts with the TRMT112 protein in mammalian cells.

<p>(A) WBSCR22 protein expression in U2OS and U2OS cell line stably expressing the E2Tag-WBSCR22 was determined by western blot using anti-WBSCR22 antibody. α-tubulin detected with anti-α-tubulin antibodies is used as a loading control. (B) Experimental scheme of the SILAC coupled co-immunoprecipitation assay performed. (C) The WBSCR22 protein interaction partners in U2OS cells stably expressing the WBSCR22 protein. H/L ratio shows the relative enrichment of the peptides of proteins pulled-down with WBSCR22 compared to mock control. (D) The WBSCR22 protein interacts with TRMT112. The WBSCR22 protein was immunoprecipitated from U2OS and U2OS-E2Tag-WBSCR22 cell lysates with an antibody against E2Tag. Proteins of the extract (Input; 10% of the lysate and pulled-down fraction (IP) were analyzed by immunoblotting with antibodies against WBSCR22, TRMT112 and C1QBP.</p

Expression of WBSCR22 mutants defective in TRMT112 binding.

<p>(A) Hela, U2OS, HepG2 and COS-7 cells were transfected with plasmids expressing EGFP, EGFP-WBSCR22, EGFP-WBSCR22-KT/AA and EGFP-WBSCR22-D117A proteins. The amount of EGFP positive cells was analyzed by flow cytometry 24, 48 and 72 hours after transfection. (B) Expression of TRMT112 and WBSCR22 proteins in HeLa cells. HeLa cells were transfected with plasmids encoding for wt WBSCR22 and its mutants with and without plasmid for TRMT112. Transfected cells were harvested 24 hours after electroporation, the lysate of 10⁵ cells was loaded on each lane and analyzed by western blot using antibody against E2Tag and α-tubulin. The non-specific signal is shown by asterisk. (C-E) Co-immunoprecipitation of WBSCR22 and TRMT112 proteins. HeLa (C) and COS-7 (E) cells were transfected with plasmids encoding for wt and mutant WBSCR22 proteins and lysed 24 hours later. (D) HeLa cells were transfected with plasmid encoding for E2Tag-TRMT112. In all cases, co-immunoprecipitation was performed using antibody against E2Tag and immunoblotting with antibodies against WBSCR22 and TRMT112. Proteins of the extract (Input; 10% of cell lysate) and pulled-down fraction (IP) were analyzed by immunoblotting.</p

WBSCR22 interaction with TRMT112 is conserved in mammals and yeast.

<p>Analysis of <i>bud23Δ</i> yeast strain complemented with human WBSCR22, its point mutants WBSCR22-D117A and WBSCR22-KT/AA and MTD containing WBSCR22 methyltransferase domain. (A) For growth dilution assays, 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 <i>bud23Δ</i> carrying plasmids for Bud23, WBSCR22, WBSCR22-D117A, WBSCR22-KT/AA, WBSCR22-MTD or vector as a control. 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.</p