RESUMO
Earlier we have demonstrated the presence of internal ribosome entry site (IRES) within tumor suppressor p53 mRNA. Here we have mapped the putative secondary structure of p53- IRES RNA using information from chemical probing and nuclease mapping experiments. Additionally, the secondary structure of the IRES element of the wild-type RNA was compared with cancer-derived silent mutant p53 RNAs. These mutations might result in the conformational alterations of p53-IRES RNAs. The results also indicate decreased IRES activities of the mutants as compared to wild-type RNA. Further, it was observed that some of the cytoplasmic trans-acting factors, critical for enhancing IRES function, were unable to bind mutant RNAs as efficiently as to wild-type. Our results suggest that hnRNP C1/C2 binds to p53-IRES and siRNA mediated partial silencing of hnRNP C1/C2 showed appreciable decrease in IRES function and consequent decrease in the level of the corresponding p53 isoform. Interestingly mutant p53 IRES showed lesser binding with hnRNP C1/C2 protein. Finally, upon doxorubicin treatment, the mutant RNAs were unable to show enhanced p53 synthesis to similar extent compared to wild type. Taken together, these observations suggest that mutations occurring in the p53 IRES might have profound implications for de-regulation of its expression and activity.
Assuntos
Mutação , Isoformas de Proteínas/biossíntese , RNA/genética , Proteína Supressora de Tumor p53/genética , Sítios de Ligação , Linhagem Celular , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Conformação de Ácido Nucleico , Mapeamento de Nucleotídeos , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência/métodos , Transfecção , Proteína Supressora de Tumor p53/biossínteseRESUMO
The p53 tumor suppressor protein plays a key role in maintaining genomic integrity. Enhanced expression of p53 during genotoxic stress is due to both increased protein stability and translational upregulation. Previous reports have shown that p53 mRNA is translated from an alternative initiation codon to produce N-terminal truncated isoform (DeltaN-p53) besides full-length p53. We have demonstrated that two internal ribosome entry sites (IRESs) regulate the translation of p53 and DeltaN-p53 in a distinct cell cycle phase-dependent manner. Here, we report that polypyrimidine tract-binding protein (PTB) is a p53 IRES interacting trans-acting factor. PTB protein binds specifically to both the p53 IRESs but with differential affinity. siRNA-mediated knockdown of PTB protein results in reduction of activity of both IRESs and also the levels of both the isoforms. It is well known that DNA-damaging agents such as doxorubicin enhance the expression of p53. Our results indicate that during doxorubicin treatment, PTB protein translocates from nucleus to the cytoplasm, probably to facilitate IRES mediated p53 translation. These observations suggest that the relative cytoplasmic abundance of PTB protein, under DNA-damaging conditions, might contribute to regulating the coordinated expression of the p53 isoforms, owing to the differential affinity of PTB binding to the two p53 IRESs.
Assuntos
Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Biossíntese de Proteínas , Proteína Supressora de Tumor p53/biossíntese , Sequência de Bases , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Dano ao DNA , Doxorrubicina/farmacologia , Humanos , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/biossíntese , Transporte Proteico/efeitos dos fármacosRESUMO
The p53 tumour suppressor protein has a crucial role in cell-cycle arrest and apoptosis. Previous reports show that the p53 messenger RNA is translated to produce an amino-terminal-deleted isoform (DeltaN-p53) from an internal initiation codon, which acts as a dominant-negative inhibitor of full-length p53. Here, we show that two internal ribosome entry sites (IRESs) mediate the translation of both full-length and DeltaN-p53 isoforms. The IRES directing the translation of full-length p53 is in the 5'-untranslated region of the mRNA, whereas the IRES mediating the translation of DeltaN-p53 extends into the protein-coding region. The two IRESs show distinct cell-cycle phase-dependent activity, with the IRES for full-length p53 being active at the G2-M transition and the IRES for DeltaN-p53 showing highest activity at the G1-S transition. These results indicate a novel translational control of p53 gene expression and activity.