RESUMO
eRF3 is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. Both bind eRF1 and stimulate its release activity in vitro. However, whether both proteins can function as termination factors in vivo has not been determined. In this study, we used short interfering RNAs to examine the effect of eRF3a and eRF3b depletion on translation termination efficiency in human cells. By measuring the readthrough at a premature nonsense codon in a reporter mRNA, we found that eRF3a silencing induced an important increase in readthrough whereas eRF3b silencing had no significant effect. We also found that eRF3a depletion reduced the intracellular level of eRF1 protein by affecting its stability. In addition, we showed that eRF3b overexpression alleviated the effect of eRF3a silencing on readthrough and on eRF1 cellular levels. These results suggest that eRF3a is the major factor acting in translation termination in mammals and clearly demonstrate that eRF3b can substitute for eRF3a in this function. Finally, our data indicate that the expression level of eRF3a controls the formation of the termination complex by modulating eRF1 protein stability.
Assuntos
Terminação Traducional da Cadeia Peptídica/fisiologia , Fatores de Terminação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Códon de Terminação/efeitos dos fármacos , Códon de Terminação/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologiaRESUMO
Enhanced stop codon readthrough is a potential treatment strategy for diseases caused by nonsense mutations. Here, we compare readthrough levels induced by three types of factors: aminoglycoside antibiotics, suppressor tRNAs, and factors decreasing translation termination efficiency. We show that the highest levels of readthrough were obtained by prolonged treatment with aminoglycosides and suppressor tRNAs, whereas prolonged depletion of release factors induced only a moderate increase in readthrough. We discuss the benefits and inconvenients of the three types of factors for their use in the therapy of diseases caused by premature stop codons.
Assuntos
Aminoglicosídeos/farmacologia , Códon/efeitos dos fármacos , Linhagem Celular , Cloranfenicol/farmacologia , Códon/genética , Humanos , Rim , Plasmídeos , RNA Mensageiro/genética , RNA Interferente PequenoRESUMO
Girolline is a 2-aminoimidazole derivative with cytotoxic activity. It affects the survival of exponentially growing leukaemic cultured cells and has a significant antitumour activity on grafted murine tumours in vivo. In vitro studies showed that girolline affected protein synthesis by interfering with the translation termination process. Here, we investigate the effect of girolline on translation termination in human cultured cells. We show that girolline neither induces an increase in translational readthrough of stop codons nor affects the polysome profile in treated cells. This suggests that girolline does not act on translation in vivo. Then, we examine the effect of girolline on cell-cycle progression and we show that girolline induces an arrest of the cell cycle at the G2 stage.