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.

The Saccharomyces cerevisiae GCN4 mRNA 5'-leader contains four upstream open reading frames (uORFs) and the CPA1 leader contains a single uORF. To determine how these uORFs control translation, we examined mRNAs containing these leaders in cell-free translation extracts to determine where ribosomes were loaded first and where they were loaded during steady-state translation. Ribosomes predominantly loaded first at GCN4 uORF1. Following its translation, but not the translation of uORF4, they efficiently reinitiated protein synthesis at Gcn4p. Adding purified eIF2 increased reinitiation at uORFs 3 or 4 and reduced reinitiation at Gcn4p. This indicates that eIF2 affects the site of reinitiation following translation of GCN4 uORF1 in vitro. In contrast, for mRNA containing the CPA1 uORF, ribosomes reached the downstream start codon by scanning past the uORF. Addition of arginine caused ribosomes that had synthesized the uORF polypeptide to stall at its termination codon, reducing loading at the downstream start codon, apparently by blocking scanning ribosomes, and not by affecting reinitiation. The GCN4 and CPA1 uORFs thus control translation in fundamentally different ways.

The Arg attenuator peptide (AAP) is an evolutionarily conserved peptide involved in Arg-specific negative translational control. It is encoded as an upstream open reading frame (uORF) in fungal mRNAs specifying the small subunit of Arg-specific carbamoyl phosphate synthetase. We examined the functions of the Saccharomyces cerevisiae CPA1 and Neurospora crassa arg-2 AAPs using translation extracts from S. cerevisiae, N. crassa, and wheat germ. Synthetic RNA containing AAP and firefly luciferase (LUC) sequences were used to program translation; analyses of LUC activity indicated that the AAPs conferred Arg-specific negative regulation in each system. The AAPs functioned either as uORFs or fused in-frame at the N terminus of LUC. Mutant AAPs lacking function in vivo did not function in vitro. Therefore, trans-acting factors conferring AAP-mediated regulation are in both fungal and plant systems. Analyses of ribosome stalling in the fungal extracts by primer extension inhibition (toeprint) assays showed that these AAPs acted similarly to stall ribosomes in the region immediately distal to the AAP coding region in response to Arg. The regulatory effect increased as the Arg concentration increased; all of the arginyl-tRNAs examined appeared maximally charged at low Arg concentrations. Therefore, AAP-mediated Arg-specific regulation appeared independent of the charging status of arginyl-tRNA.

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