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Edited by Bert Vogelstein, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD (received for review April 10, 2003)
Article Figures & SI
Figures
- Fig. 1.
Expression of p53 in RKO cells irradiated with UVC. (A) Cells were exposed to either 15 or 30 J/m2 or left unirradiated (-), and whole-cell lysates were prepared for Western blotting at the times indicated. (B) Northern blot analysis of p53 expression 4 h after exposure to the indicated doses of UVC. Relative p53 abundance was calculated by densitometry and is expressed as fold increase relative to p53 levels in unirradiated cells. (C) Half-life of the p53 mRNA was calculated by addition of actinomycin D (ActD, 2 μg/ml) to RKO cells 4 h after either UVC irradiation (15 J/m2) or no treatment (untreated), as described (11). Relative p53 mRNA abundance is expressed as fold increase relative to p53 mRNA levels in unirradiated cells. (D) Total, cytoplasmic (Cytopl.), nuclear, and polysomal fractions were prepared for Northern blot analysis 4 h after exposure to UVC (15 J/m2); 18S signals reveal even loading of samples.
- Fig. 2.
(A) Western blot analysis of p53 expression in RKO whole-cell lysates prepared at the times indicated after treatment with lactacystin (5 μM), UVC (15 J/m2), or a combination of both. Signals were quantitated by densitometry (graph). (B) Newly translated p53 was measured by incubating cells with l-[35S]methionine and l-[35S]cysteine for 20 min, followed by immunoprecipitation by using either anti-p53 antibody or IgG1, resolving immunoprecipitated samples by SDS/PAGE, and transferring for visualization of signals by using a PhosphorImager.
- Fig. 3.
Binding of cytoplasmic proteins to the p53 3′ UTR is linked to p53 translational up-regulation. (Upper) Schematic of p53 mRNA, depicting U-rich and AU-rich sequences (gray), as well as transcripts (CR and 3′ UTR) used for analysis. (A) REMSA (11) by using either CR or 3′ UTR p53 radiolabeled transcripts and cytoplasmic lysates prepared 4 and 6 h after treatment of RKO cells with either 15 or 30 J/m2 UVC. (B) Plasmids pGL3-Luc and pGL3-Luc-p53(3′UTR) were used in transient transfections; 24 h after transfection, cells were either irradiated (15 J/m2) or left untreated, and the relative luciferase activity was calculated 6, 12, and 24 h later.
- Fig. 4.
HuR binds to the p53 3′ UTR in a UVC-dependent manner. (A) REMSA supershift using either IgG1 or an anti-HuR antibody (11) (Left), or antibodies recognizing the RNA-binding proteins shown (Right). f, free probe, digested with RNase T1 but left without incubating with cytoplasmic lysates; arrowhead, supershift of HuR-containing complexes. (B) Detection of HuR after pull-down by using biotinylated p53 transcripts CR and 3′ UTR (described in Fig. 3 Upper). After incubations with proteins present in cytoplasmic, cytosolic, or polysomal fractions of cells that were either left untreated or treated with 15 J/m2 UVC, the presence of HuR and β-actin was detected by Western blotting. (C) IP by using either anti-HuR antibody or IgG1 under conditions that preserve the association of RNA-binding proteins with target mRNAs was followed by RT-PCR analysis to detect endogenous p53 mRNA; PCR products were resolved by electrophoresis in 1.5% agarose gels stained with ethidium bromide. (D) Subcellular localization of HuR in cytoplasmic (Cytopl.), cytosolic (Cytosol), and polysomal (Polysome) fractions prepared from cells that were either left untreated or UVC-treated.
- Fig. 5.
Modulation of HuR levels affects p53 translation and steady-state levels. (A) Western blot analysis of HuR and p53 expression levels in RKO cells that had been stably transfected with either an insert less plasmid (zeo) or an HuR-expressing plasmid (S11). (B) RKO cells were transiently transfected with either a control (Ctrl) siRNA or siRNA directed to HuR (HuR4), and exposed to 15 J/m2 UVC 48 h later. HuR expression levels were analyzed by Western blotting using whole-cell lysates that were prepared 4 h after UVC. (C) Newly translated p53 was assessed as explained in the legend of Fig. 2B. Forty-eight hours after transfection with either control or HuR4 siRNAs, RKO cultures were irradiated or left untreated; 4 h later, they were incubated with l-[35S]methionine and l-[35S]cysteine for 20 min. Lysates were prepared and immunoprecipitated by using either anti-p53 antibody or IgG1, then processed as described in Fig. 2B.
RNA-binding protein HuR enhances p53 translation in response to ultraviolet light irradiation
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