RESUMEN
The tumor suppressor p53 protein is expressed at low levels under normal conditions. The subcellular localization and functional activation of p53 are influenced by diverse stress signals. p53 in cytoplasm exerts intrinsic 3'â5' exonuclease activity with various RNA and DNA substrates. ssRNAs containing an adenosine and uridine-rich (ARE) element are permissive targets for p53-mediated degradation. The analysis of the exonuclease activity in cytoplasm with activated p53 induced by various drug treatments or following γ-irradiation revealed that the expression of p53 exonuclease activity in response to stress signals is heterogeneous. Various genotoxic and non-genotoxic agents upregulate p53 yet have different effects on expression of exonuclease activity with ARE RNA but not with DNA substrate. Ribonuclease activity is enhanced in cytoplasmic extracts of HCT116 (p53+/+) cells exposed to γ-irradiation or treated by the non-genotoxic drug AS101 but decreased following treatment by genotoxic (e.g., doxorubicin) or non-genotoxic (e.g., DFMO) agents, thus indicating that p53 exonuclease activity is dependent on the specific stress and nature of the substrate. Apparently, the disparity in expression of p53 ribonuclease activity after each treatment is attributable to the different post-treatment response and to two posttranscriptional events: the interaction of RNA-binding HuR protein with ARE RNA protects the substrate from degradation by p53 and/or decrease in p53 ARE RNA binding capacity due to phosphorylation at Ser392 leads to reduction in p5 ribonuclease activity. Our results provide new insights into p53 exonuclease function and into the mechanisms behind the regulation ARE-RNA degradation by p53 under different cellular conditions.
Asunto(s)
Citoplasma/enzimología , Exonucleasas/metabolismo , Estabilidad del ARN , ARN/metabolismo , Ribonucleasas/metabolismo , Estrés Fisiológico , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales , Citoplasma/efectos de la radiación , Doxorrubicina/farmacología , Proteínas ELAV/metabolismo , Eflornitina/farmacología , Etilenos/farmacología , Etopósido/farmacología , Exonucleasas/genética , Rayos gamma , Humanos , Inhibidores de la Ornitina Descarboxilasa , Fosforilación , ARN/química , Ribonucleasas/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Double-stranded RNA (dsRNA) is a biologically active molecule that plays important roles in normal cell growth and function. Accordingly, the cell uses multiple mechanisms to control its level. The tumor suppressor protein p53 possesses intrinsic 3'â5' exonuclease activity. The aim of the present study was to elucidate the degradation of dsRNA by the exonuclease activity of p53. The results show that recombinant, purified wtp53 and endogenous protein in cytoplasmic fractions of cells remove nucleotides from 3'-ends of dsRNA. Several lines of evidence support a connection between p53 and dsRNase activity in cytoplasm: (1) this activity parallels the status of endogenous cytoplasmic p53; (2) the endogenous exonuclease displays a similar dsRNA excision profile characteristic for purified wtp53; (3) cytoplasmic fractions of HCT116(p53+/+) cells exert higher levels of exonuclease activity compared to those of HCT116(p53-/-) cells; (4) transfection of the wtp53, but not exonuclease-deficient mutant p53-R175H, into HCT116 (p53-/-) cells induced high levels of dsRNase activity in cytoplasm; (5) the accumulation of p53 in cytoplasm following the γ-irradiation stress stimuli correlates with the increase in the excision of dsRNA and (6) the dsRNA forms a complex with a protein that can be disrupted by an anti-p53 antibody. Our data suggest that the degradation of dsRNA by p53 protein may direct either the complete degradation of and decrease in the level of dsRNA or incomplete degradation and the generation of short dsRNA products. The possible roles of p53 dsRNase activity in cytoplasm in the inhibition of translation and induction of cell apoptosis, is discussed.