Fig. 2.

Wig-1 knockdown decreases p53 mRNA stability through increased deadenylation. (A) Northern blot analysis shows decreased p53 mRNA levels after treatment with Wig-1 siRNA in U2OS cells. c and wA (Wig-1 siRNA), both from Ambion. (B) MCF7 cells were treated with siRNA (c and w1) and subsequently with the transcriptional inhibitor actinomycin (Act) D and harvested at 6 or 12 h. Total RNA was analyzed by Northern blotting followed by quantification using densitometry (NB) or analyzed by real-time PCR (qRT). The p53 levels were normalized to GAPDH RNA. The figure shows the mean of three independent experiments. Bars are shown with SEM (P = 0.0005 and P = 0.011 at 6 h, and P = 0.0001 and P = 0.0002 at 12 h for the Northern blotting and the real-time data, respectively). (C) Analysis of the length of the p53 poly(A) tail on Wig-1 knockdown. We prepared total RNA from siRNA-treated cells, added an adapter to the 3′ end of the mRNA, and performed cDNA synthesis followed by PCR of the end of the p53 3′ UTR (see Materials and Methods; Fig. S2f). PCR products were separated by gel electrophoresis, blotted to a membrane, and detected by using radiolabeled probes. Wig-1 knockdown resulted in decreased size of the amplified product, showing that absence of Wig-1 leads to enhanced deadenylation of the p53 mRNA. (D) Immunofluorescence staining for Wig-1 in U2OS cells demonstrates both nuclear and cytoplasmic localization of Wig-1. (E) HCT116 p53+/+ cells untreated or treated with camptothecin were fractionated into nucleus and cytoplasm and 30 μg of protein from each fraction (corresponding to ≈1% of the cytoplasmic fractions and 20% of the nuclear fractions) was analyzed by Western blotting alongside total lysate. The figure shows an increase in Wig-1 levels in both compartments after camptothecin treatment. Arrows indicate the main Wig-1 species. Lamin A/C is a nuclear marker and Hsp90 a cytoplasmic marker.