Leaky termination at premature stop codons antagonizes nonsense-mediated mRNA decay in S. cerevisiae

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Abstract
The Nonsense-Mediated mRNA Decay (NMD) pathway mediates the rapid degradation of mRNAs that contain premature stop mutations in eukaryotic organisms. It was recently shown that mutations in three yeast genes that encode proteins involved in the NMD process, UPF1, UPF2, and UPF3, also reduce the efficiency of translation termination. In the current study, we compared the efficiency of translation termination in a upf1Δ strain and a [PSI+] strain using a collection of translation termination reporter constructs. The [PSI+] state is caused by a prion form of the polypeptide chain release factor eRF3 that limits its availability to participate in translation termination. In contrast, the mechanism by which Upf1p influences translation termination is poorly understood. The efficiency of translation termination is primarily determined by a tetranucleotide termination signal consisting of the stop codon and the first nucleotide immediately 3′ of the stop codon. We found that the upf1Δ mutation, like the [PSI+] state, decreases the efficiency of translation termination over a broad range of tetranucleotide termination signals in a unique, context-dependent manner. These results suggest that Upf1p may associate with the termination complex prior to polypeptide chain release. We also found that the increase in readthrough observed in a [PSI+]/upf1Δ strain was larger than the readthrough observed in strains carrying either defect alone, indicating that the upf1Δ mutation and the [PSI+] state influence the termination process in distinct ways. Finally, our analysis revealed that the mRNA destabilization associated with NMD could be separated into two distinct forms that correlated with the extent the premature stop codon was suppressed. The minor component of NMD was a 25% decrease in mRNA levels observed when readthrough was ≥0.5%, while the major component was represented by a larger decrease in mRNA abundance that was observed only when readthrough was ≤0.5%. This low threshold for the onset of the major component of NMD indicates that mRNA surveillance is an ongoing process that occurs throughout the lifetime of an mRNA.