Pseudouridine-mediated stop codon readthrough in S. cerevisiae is sequence context-independent

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Stop codons normally terminate translation.
• Pseudouridylation of uridine in stop codons can lead to ribosome readthrough.
• The sequence context dependency of this pseudouridine-mediated readthrough is not well understood.

Purpose of the Study:

• To investigate whether pseudouridine-mediated stop codon readthrough is dependent on mRNA sequence context.
• To determine the role of upstream and surrounding nucleotides in pseudouridine-mediated readthrough.
• To assess the effect of targeted pseudouridylation on nonsense-mediated mRNA decay (NMD).

Main Methods:

• Utilized a yeast system with reporter mRNAs.
• Introduced premature termination codons (PTCs) at various positions with different sequence contexts.
• Employed mutational and selection/screen analyses.
• Assessed ribosome readthrough levels and NMD suppression.

Main Results:

• Similar levels of ribosome readthrough were observed when pseudouridylated stop codons were placed in different sequence contexts.
• Upstream and surrounding nucleotides played a minimal role in pseudouridine-mediated readthrough.
• Targeted pseudouridylation of PTCs did not suppress NMD in the yeast system.

Conclusions:

• Pseudouridine-mediated nonsense suppression is a sequence context-independent translational event.
• This mechanism differs from previously identified rare stop codon readthrough events.
• Pseudouridylation of stop codons does not inhibit NMD in yeast.