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.
D020413:3' Untranslated Regions; D000199:Actins / Q000378:metabolism; D000263:Adenylate Kinase / Q000378:metabolism; D000954:Antigens, Surface; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002292:Carcinoma, Renal Cell / Q000378:metabolism*; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007680:Kidney Neoplasms / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D010957:Plasmids / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D015854:Up-Regulation; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D020413:3' Untranslated Regions; D000199:Actins; D000954:Antigens, Surface; D018076:DNA, Complementary; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D012313:RNA; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D000263:Adenylate Kinase; C497446:VHL protein, human