Ribosome occupancy of the yeast CPA1 upstream open reading frame termination codon modulates nonsense-mediated mRNA decay.

Saccharomyces cerevisiae CPA1 mRNA contains an upstream open reading frame (uORF) encoding the arginine attenuator peptide (AAP). Negative translational regulation of CPA1 occurs when the nascent AAP responds to arginine (Arg) by stalling ribosomes at the uORF termination codon. CPA1 expression is also controlled by nonsense-mediated mRNA decay (NMD). Using wild-type and decay-defective strains expressing CPA1-LUC, we determined how this uORF contributes to NMD control. Arg addition to media rapidly destabilized the CPA1 transcript in wild-type but not upf1delta cells. The wild-type uORF exerted translational control and induced NMD of CPA1-LUC; the mutated D13N uORF, which eliminates stalling and regulation, did not. Thus, regulation by NMD was not governed simply by ribosomes encountering the uORF terminator but appeared dependent on the AAP's ribosome-stalling ability. Improving the D13N uORF initiation context also promoted NMD. Hence, NMD appears to be triggered by increased ribosomal occupancy of the uORF termination codon.