Influence of the RNA-binding protein HuR in pVHL-regulated p53 expression in renal carcinoma cells.
A recent analysis of gene expression in renal cell carcinoma cells led to the identification of mRNAs whose translation was dependent on the presence of the von Hippel-Lindau (VHL) tumor suppressor gene product, pVHL. Here, we investigate the finding that pVHL-expressing RCC cells (VHL(+)) exhibited elevated levels of polysome-associated p53 ... mRNA and increased p53 protein levels compared with VHL-defective (VHL(-)) cells. Our findings indicate that p53 translation is specifically heightened in VHL(+) cells, given that (i) p53 mRNA abundance in VHL(+) and VHL(-) cells was comparable, (ii) p53 degradation did not significantly influence p53 expression, and (iii) p53 synthesis was markedly induced in VHL(+) cells. Electrophoretic mobility shift and immunoprecipitation assays to detect endogenous and radiolabeled p53 transcripts revealed that the RNA-binding protein HuR, previously shown to regulate mRNA turnover and translation, was capable of binding to the 3' untranslated region of the p53 mRNA in a VHL-dependent fashion. Interestingly, while whole-cell levels of HuR in VHL(+) and VHL(-) cells were comparable, HuR was markedly more abundant in the cytoplasmic and polysome-associated fractions of VHL(+) cells. In keeping with earlier reports, the elevated cytoplasmic HuR in VHL(+) cells was likely due to the reduced AMP-activated kinase activity in these cells. Demonstration that HuR indeed contributed to the increased expression of p53 in VHL(+) cells was obtained through use of RNA interference, which effectively reduced HuR expression and in turn caused marked decreases in p53 translation and p53 abundance. Taken together, our findings support a role for pVHL in elevating p53 expression, implicate HuR in enhancing VHL-mediated p53 translation, and suggest that VHL-mediated p53 upregulation may contribute to pVHL's tumor suppressive functions in renal cell carcinoma.
Mesh Terms:
3' Untranslated Regions, Actins, Adenylate Kinase, Antigens, Surface, Base Sequence, Blotting, Northern, Blotting, Western, Carcinoma, Renal Cell, Cell Line, Tumor, Cytoplasm, DNA, Complementary, Gene Expression Regulation, Neoplastic, Humans, Kidney Neoplasms, Microscopy, Fluorescence, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Plasmids, Polyribosomes, Precipitin Tests, Protein Binding, Protein Biosynthesis, RNA, RNA Interference, RNA, Messenger, RNA, Small Interfering, RNA-Binding Proteins, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, Transfection, Tumor Suppressor Protein p53, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases, Up-Regulation, Von Hippel-Lindau Tumor Suppressor Protein
3' Untranslated Regions, Actins, Adenylate Kinase, Antigens, Surface, Base Sequence, Blotting, Northern, Blotting, Western, Carcinoma, Renal Cell, Cell Line, Tumor, Cytoplasm, DNA, Complementary, Gene Expression Regulation, Neoplastic, Humans, Kidney Neoplasms, Microscopy, Fluorescence, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Plasmids, Polyribosomes, Precipitin Tests, Protein Binding, Protein Biosynthesis, RNA, RNA Interference, RNA, Messenger, RNA, Small Interfering, RNA-Binding Proteins, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, Transfection, Tumor Suppressor Protein p53, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases, Up-Regulation, Von Hippel-Lindau Tumor Suppressor Protein
Mol. Cell. Biol.
Date: Oct. 01, 2003
PubMed ID: 14517280
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