FIGURE 2.

WDR3 is required for rRNA processing. A, U2OS cells were transfected with mock, control siRNA (siNEG), or two siRNA oligonucleotides targeting WDR3, siWDR3 A, and siWDR3 B. WDR3 protein levels were analyzed by Western blot analysis 72 h post-siRNA transfection using commercially available WDR3 antibody. β-Actin was used as a loading control. Data shown indicate WDR3 protein levels in cells used in C and represent one of three independent experiments with similar results. B, schematic diagram representing the primary 47 S rRNA transcript and mammalian rRNA processing pathways, with the position of hybridization probes shown. C, total RNA from U2OS cells transfected with mock, control siRNA (siNEG), or siRNA targeting WDR3 was analyzed by Northern blotting for rRNA species using probes specific for human ITS-1, ITS-2, and 28 and 18 S rRNA. Ratios of the 18/28 S rRNA are shown. Results represent one of two independent experiments with similar results. D, total RNA from MCF-7 cells transfected with mock, control siRNA (siNEG), or siRNA targeting WDR3 was analyzed by Northern blotting 72 h post-transfection using 28 and 18 S rRNA probes. The ratios of the 18/28 S rRNA are shown. The graph represents fold change in 18/28 S rRNA levels relative to mock-treated cells from three independent experiments (p = 0.05, Student's t test). E, total RNA from siRNA-transfected U2OS cells was analyzed by S1 nuclease protection assay using a probe complementary to the first 40 nucleotides of 47 S pre-RNA, and total protein was analyzed for β-actin expression by Western blotting (top panel). pre-rRNA and β-actin signals were quantified from triplicate samples using Aida software, and the levels of pre-rRNA were normalized to β-actin levels and expressed as a percentage from the highest signal (set at 100%) (bottom panel).