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 TNFα. 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 TNFα 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 TNFα 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.

A synthetic library of RNA control modules for predictable tuning of gene expression in yeast

“…The set of synthetic Rnt1p substrates developed in this work represents the first engineered library of transcript stability control modules in the yeast S. cerevisiae . Although other posttranscriptional regulatory elements, such as internal ribosome entry sites (IRESes) and AU‐rich elements, have been applied to regulate heterologous gene expression in yeast, such genetic elements have exhibited substantial variability in activity and have not been engineered as synthetic libraries of control modules exhibiting a wide range of activities (Vasudevan and Peltz, 2001; Zhou et al , 2001; Lautz et al , 2010). In addition, a library of short synthetic IRESes that act through translation initiation was previously developed for yeast (Zhou et al , 2003).…”

“…RNA‐based control modules based on posttranscriptional mechanisms may offer an advantage in that they can be coupled to any promoter of choice, providing for enhanced control strategies and finer resolution tuning of protein expression levels. Although posttranscriptional control elements, such as internal ribosome entry sites and AU‐rich elements, have been applied to regulate heterologous gene expression in yeast (Vasudevan and Peltz, 2001; Zhou et al , 2001; Lautz et al , 2010), these control elements have exhibited substantial variability in activity and have not been engineered as synthetic libraries exhibiting a wide range of predictable gene regulatory activities. RNase III enzymes are a class of enzymes that cleave double‐stranded RNA. The S. cerevisiae RNase III enzyme, Rnt1p, exhibits a number of unique features that allow it to recognize very specific RNA hairpin substrates that harbor a consensus AGNN tetraloop sequence.…”

Pou1f1, the key transcription factor related to somatic growth in tilapia (Orechromis niloticus), is regulated by two independent post-transcriptional regulation mechanisms

“…Furthermore, mice carrying an endogenous deletion of the AU-Rich region of the tnfα gene suffer from inflammation and develop a Crohn's disease-like phenotype [12]. The function of the Class II AU-Rich element is conserved through eukaryotes [26],…”