WDR55 modulates nucleolar rRNA production and cell cycle progression.

<p>(A) Intracellular localization of transfected EGFP, EGFP-WDR55WT, and EGFP-WDR55hkc (green) along with co-transfected t-HcRed1-fibrillarin in 293T cells. Single fluorescence images and merged images are shown. Rightmost panels show images of single cells isolated from stage-19 embryos that were administered at 1-cell stage with mRNAs of indicated genes. (B) Localization of endogenous WDR55 in NIH3T3 cells identified by antibody staining. Single fluorescence images and merged images of WDR55 (green) and fibrillarin (red, top panels) or B23 (red, bottom panels) are shown. (C) Western blotting of whole cell lysates of control or WDR55-siRNA-transfected NIH3T3 cells at 44 hours after transfection using anti-calnexin or anti-WDR55 antibody. (D) Detection of rRNA processing intermediates by Northern blot hybridization with 5.8S rRNA probe of total RNA (0.5 µg per lane) isolated from untransfected, control-siRNA-transfected, and WDR55-siRNA-transfected NIH3T3 cells. Asterisks indicate accumulation of processing intermediates in NIH3T3 cells transfected with WDR55-siRNA. An image of methylene blue (MB) staining is also shown (left). The amount of housekeeping <i>glyceraldehyde-3-phosphate dehydrogenase</i> mRNA measured by quantitative RT-PCR was not significantly different among same-weight total cellular RNAs isolated from untransfected, control-siRNA-transfected, and WDR55-siRNA-transfected cells. Along with the data showing that the intensities of the bands for 28S, 18S, and 5.8S rRNAs were comparable among these three groups of cells, these results suggest that the total amounts of rRNAs were comparable among these three groups of cells. (E) Immunoprecipitation (IP) of U2OS cell lysates using anti-WDR12, anti-Pes1, and anti-Bop1 antibodies, or normal rat IgG. 2.5% of input lysate before IP was also electrophoresed. Western blot (WB) detection is shown for Pes1 (top) and WDR55 (bottom). (F) Quantitative RT-PCR analysis of indicated genes in control (open bars) and WDR55-siRNA-transfected (closed bars) NIH3T3 cells. Expression levels in control cells were normalized to 1. Results represent averages and standard errors of three independent measurements. Asterisks, p<0.05. (G) Cell cycle analysis of control (left) and WDR55-siRNA-transfected NIH3T3 cells by BrdU and 7-AAD staining. Numbers indicate frequency of cells in indicated squares. Lower left box, upper box, and lower right box show cells in G1 phase, S phase, and G2/M phase, respectively. Representative results of three independent experiments are shown.