Analysis of cotranslational polyamine sensing during decoding of ODC antizyme mRNA

Abstract

Polyamines are essential poly-cations with vital functions in all cellular systems. Their levels are controlled by intricate regulatory feedback mechanisms. Abnormally high levels of polyamines have been linked to cancer. A rate-limiting enzyme in the biosynthesis of polyamines in fungi and higher eukaryotes is ornithinedecarboxylase (ODC). Its levels are largely controlled posttranslationally via ubiquitin-independent degradation mediated by ODC antizyme (OAZ). The latter is a critical polyamine sensor in a feedback control mechanism that adjusts cellular polyamine levels. Here, we describe an approach employing quantitative western blot analyses that provides in vivo evidence for cotranslational polyamine-sensing by nascent OAZ in yeast. In addition, we describe an in vitro method to detect polyamine binding by antizyme.