RESUMO
p53 is a well known tumor suppressor protein that plays a critical role in cell cycle arrest and apoptosis. It has several isoforms which are produced by transcriptional and posttranscriptional regulatory mechanisms. p53 mRNA has been demonstrated to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform ΔN-p53 by the use of alternative translation initiation sites. The mechanism of translation regulation of these two isoforms was further elucidated by the discovery of IRES elements in the p53 mRNA. These two IRESs were shown to regulate the translation of p53 and ΔN-p53 in a distinct cell-cycle phase-dependent manner. This review focuses on the current understanding of the regulation of p53 IRES mediated translation and the role of cis and trans acting factors that influence expression of p53 isoforms.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , Ribossomos/genéticaRESUMO
p53 mRNA has been shown to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which modulates the functions of FL-p53 and also has independent functions. Previously, we have shown that translation of p53 and ΔN-p53 can be initiated at Internal Ribosome Entry Sites (IRES). These two IRESs were shown to regulate the translation of p53 and ΔN-p53 in a distinct cell-cycle phase-dependent manner. Earlier observations from our laboratory also suggest that the structural integrity of the p53 RNA is critical for IRES function and is compromised by mutations that affect the structure as well as RNA protein interactions. In the current study, using RNA affinity approach we have identified Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) as novel ITAFs for p53 IRESs. We have showed that the purified Annexin A2 and PSF proteins specifically bind to p53 IRES elements. Interestingly, in the presence of calcium ions Annexin A2 showed increased binding with p53 IRES. Immunopulldown experiments suggest that these two proteins associate with p53 mRNA ex vivo as well. Partial knockdown of Annexin A2 and PSF showed decrease in p53 IRES activity and reduced levels of both the p53 isoforms. More importantly the interplay between Annexin A2, PSF and PTB proteins for binding to p53mRNA appears to play a crucial role in IRES function. Taken together, our observations suggest pivotal role of two new trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.
Assuntos
Anexina A2/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Anexina A2/genética , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fator de Processamento Associado a PTB , Ligação Proteica , Mapeamento de Interação de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
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.
