Arabidopsis Polyamine oxidase-2 uORF is required for downstream translational regulation

In eukaryotic mRNAs, small upstream open reading frames (uORFs) located in the 5'-untranslated region control the translation of the downstream main ORF. Polyamine oxidase (PAO) enzymes catalyze the oxidation of higher polyamines such as spermidine and spermine, and therefore contribute to the maintenance of intracellular polyamine content and to the regulation of physiological processes through their catabolic products. Recently, we reported that the Arabidopsis thaliana Polyamine Oxidase 2 (AtPAO2) is post-transcriptionally regulated by its 5'-UTR region through an uORF. In the present study, we analyzed whether the translation of the uORF is needed for the translational repression of the main ORF, and whether the inactivation of the uORF had an effect on the translational control mediated by polyamines. To this aim, we generated diverse single mutations in the uORF sequence; these mutant 5'-UTRs were fused to the GUS reporter gene, and tested in onion monolayer cells and A. thaliana transgenic seedlings. Removal of the start codon or introduction of a premature stop codon in the AtPAO2 uORF sequence abolished the negative regulation of the GUS expression exerted by the wild-type AtPAO2 uORF. An artificial uORF (32 amino acids in length) generated by the addition of a single nucleotide in AtPAO2 uORF proved to be less repressive than the wild-type uORF. Thus, our findings suggest that translation of the AtPAO2 uORF is necessary for the translational repression of the main ORF.