Ribosome stalling at an upstream open reading frame regulates nonsense-mediated mRNA decay.
Anthony Gaba (1), Allan Jacobson (2), Matthew S. Sachs (1)
(1) Environ. Biomolec. Systems, Oregon Health & Science Univ, 20000 NW Walker Road, Beaverton, OR, 97006, US;
(2) Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655-0122
The yeast CPA1 mRNA contains an upstream open reading frame (uORF) specifying the arginine attenuator peptide (AAP), whose translation is critical for arginine-specific negative regulation of gene expression. In cell free systems, regulation depends on the AAP's ability to act as a nascent peptide to stall ribosomes at the uORF termination codon in response to arginine. An AAP missense mutation (D13N) eliminates stalling and regulation. AAP-mediated ribosome stalling appears to block the access of scanning ribosomes to the downstream CPA1 start codon. CPA1 expression is also affected by nonsense-mediated mRNA decay (NMD). To determine how the CPA1 uORF contributes to NMD-mediated control of CPA1 expression, we constructed isogenic nmd2delta and NMD+ strains expressing CPA1-LUC chimeric transcripts. Levels of luciferase enzyme activity and RNA were measured. Our results indicate that, when the CPA1 uORF is in its normal poor initiation context, translation of the wild-type uORF induced NMD of the CPA1-LUC transcript, but the mutated D13N uORF did not. When the uORF initiation context was improved, the D13N uORF also triggered NMD. CPA1 regulation by NMD thus normally appears dependent on the AAP's ability to stall ribosomes in the mRNA 5'-leader at the uORF termination codon. Stalling at that site may enable efficient recruitment of NMD factors. Improving the uORF initiation context may be an alternative way to recruit ribosomes to that site for triggering NMD.
Return to YGM 2004 Home at SGD