The human c‐fos and TNFα AU‐rich elements show different effects on mRNA abundance and protein expression depending on the reporter in the yeast Pichia pastoris

Abstract

AU-rich elements (AREs) are located in the 3' untranslated region (3' UTR) of their host genes and tightly regulate mRNA degradation and expression. Examples for this kind of regulation are the human proto-oncogene c-fos and the cytokine TNFalpha. Despite large effort in this field, the exact mechanism of ARE-mediated mRNA turnover remains unclear. In this work we analysed the effects of c-fos- and TNFalpha AREs on mRNA abundance and protein expression of selected human cDNAs in the yeast Pichia pastoris. This yeast is exceedingly well known for its excellent protein production capacity; however, ARE-like mechanisms have not been studied in this yeast to date. Interestingly, we observed both stabilizing and destabilizing effects of the c-fos ARE, whereas the TNFalpha ARE has a destabilizing or expression-reducing function in all tested cDNAs. Based on this observation, we introduced a number of single-point mutations upstream of the introduced c-fos ARE into the 3' UTR of a single cDNA in order to demonstrate the importance of ARE-flanking sequences for their own regulation. In conclusion, we illustrate that the analysis of ARE-mediated effects on mRNA abundance and protein expression of a reporter depends on the sequence of the reporter itself as well as the ARE-surrounding sequences within the 3' UTR. For this reason, we question whether already established reporter constructs in other cellular systems display the true type of regulation of the tested AREs for its original host gene. Finally, we propose that AREs should be analysed in their native sequence context.