BACKGROUND: Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual +1 translational frameshift mechanism that responds to polyamine levels. RESULTS: HEK293 cells were depleted of polyamines by treatment with an ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and grown in the presence or absence of exogenous polyamines prior to the analysis of ribosomal frameshifting levels. Results obtained using an optimized dual luciferase assay system reveal a 10-fold dynamic range of frameshifting, which correlates positively with polyamine addition. Polyamine addition to cells, which have not been pre-treated with DFMO, also resulted in an increase in antizyme frameshifting but to a lesser degree (1.3 to 1.5-fold). In addition, the constructs with the 3' deletion were more responsive to stimulation by polyamine addition than those with the 5' deletion. CONCLUSIONS: The observed regulation of antizyme frameshifting demonstrates the efficiency of a polyamine homeostatic mechanism, and illustrates the utility of a quantifiable cell-based assay for the analysis of polyamines or their analogues on translational frameshifting.
BACKGROUND: Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual +1 translational frameshift mechanism that responds to polyamine levels. RESULTS: HEK293 cells were depleted of polyamines by treatment with an ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and grown in the presence or absence of exogenous polyamines prior to the analysis of ribosomal frameshifting levels. Results obtained using an optimized dual luciferase assay system reveal a 10-fold dynamic range of frameshifting, which correlates positively with polyamine addition. Polyamine addition to cells, which have not been pre-treated with DFMO, also resulted in an increase in antizyme frameshifting but to a lesser degree (1.3 to 1.5-fold). In addition, the constructs with the 3' deletion were more responsive to stimulation by polyamine addition than those with the 5' deletion. CONCLUSIONS: The observed regulation of antizyme frameshifting demonstrates the efficiency of a polyamine homeostatic mechanism, and illustrates the utility of a quantifiable cell-based assay for the analysis of polyamines or their analogues on translational frameshifting.
Authors: John F Atkins; Gary Loughran; Pramod R Bhatt; Andrew E Firth; Pavel V Baranov Journal: Nucleic Acids Res Date: 2016-07-19 Impact factor: 16.971