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

Area of Science:

• Molecular Biology
• Gene Regulation
• Yeast Genetics

Background:

• Saccharomyces cerevisiae CPA1 mRNA features an upstream open reading frame (uORF) encoding the arginine attenuator peptide (AAP).
• CPA1 expression is subject to translational repression by AAP-mediated ribosome stalling at the uORF in response to arginine (Arg).
• Nonsense-mediated mRNA decay (NMD) also regulates CPA1 mRNA levels.

Purpose of the Study:

• To investigate the role of the CPA1 uORF in controlling NMD.
• To determine if ribosome stalling at the uORF termination codon is essential for NMD induction.
• To elucidate the mechanism by which the uORF influences CPA1 mRNA stability.

Main Methods:

• Utilized wild-type and decay-defective (upf1delta) Saccharomyces cerevisiae strains.
• Employed CPA1-LUC reporter constructs to monitor gene expression and mRNA stability.
• Manipulated arginine levels in growth media.
• Introduced mutations in the uORF (D13N) to abolish ribosome stalling.

Main Results:

• Arginine addition rapidly destabilized CPA1 mRNA in wild-type cells but not in upf1delta cells.
• A functional wild-type uORF induced NMD of CPA1-LUC, whereas a non-stalling D13N uORF mutant did not.
• Enhancing the initiation context of the D13N uORF promoted NMD, suggesting a role for ribosomal occupancy.

Conclusions:

• NMD regulation of CPA1 mRNA is dependent on the ribosome-stalling ability of the AAP within the uORF.
• NMD is triggered by increased ribosomal occupancy at the uORF termination codon, not merely by ribosome encounter.
• The interplay between translation and mRNA decay pathways fine-tunes gene expression in response to nutrient availability.