Misdecoding of rare CGA codon by translation termination factors, eRF1/eRF3, suggests novel class of ribosome rescue pathway in S. cerevisiae

Abstract

The CGA arginine codon is a rare codon in Saccharomyces cerevisiae. Thus, full‐length mature protein synthesis from reporter genes with internal CGA codon repeats are markedly reduced, and the reporters, instead, produce short‐sized polypeptides via an unknown mechanism. Considering the product size and similar properties between CGA sense and UGA stop codons, we hypothesized that eukaryote polypeptide‐chain release factor complex eRF1/eRF3 catalyses polypeptide release at CGA repeats. Herein, we performed a series of analyses and report that the CGA codon can be, to a certain extent, decoded as a stop codon in yeast. This also raises an intriguing possibility that translation termination factors eRF1/eRF3 rescue ribosomes stalled at CGA codons, releasing premature polypeptides, and competing with canonical tRNAICG to the CGA codon. Our results suggest an alternative ribosomal rescue pathway in eukaryotes. The present results suggest that misdecoding of low efficient codons may play a novel role in global translation regulation in S. cerevisiae.

CTELS: A Cell-Free System for the Analysis of Translation Termination Rate

“…We used ~25–50 ng/μL of recombinant factors, while ~50 ng/μL of the endogenous eRF1 was present in the Krebs-2 system, according to a previous estimation [ 23 ], and a similar eRF1 concentration (~85 ng/μL) was reported for HeLa cells [ 103 ]. It is likely that in the case of wt eRF1, its non-specific binding to elongating ribosomes caused premature termination, in accordance with the previous findings [ 102 , 104 ].…”

“…Previously, a decoding of the read-through-prone UGA-C stop codon was also shown to occur very slowly in E. coli [119], which corresponds well to our finding and suggests the universality of the stalling-induced model of nonsense suppression. Thus, the mechanism of stop codon recoding could be the reversed side of the situation when rare sense codons are recoded by eRF1 (shown in [102,104] and also observed here, see Figure 2d and Figure S2), both are based on transient ribosome stalling followed by its inappropriate resolution. appearance was almost as long as when a transcript with the zero-length 3′ UTR was used.…”

“…We further showed that release factor eRF1, as well as its catalytically inactive mutant eRF1(AGQ), when taken in elevated concentration, can perturb and significantly slow elongation, most likely by interfering with the eEF1–tRNA binding to the ribosome ( Figure 2 d and Figure S2 ). The ability of release factors to bind non-specifically to near-cognate codons [ 101 ] and to induce premature termination at rare codons [ 102 , 104 ] was shown previously by other methods, although this fact is not widely accepted in the field of protein synthesis research. Nevertheless, it is well-known that phenotypes of mutation in eRF1 and eRF3 genes, as well as their overexpression, are pleiotropic and are sometimes unlinked with translation termination defects [ 111 , 112 ].…”

Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

“…Therefore, special care will be required when analyzing CGA. However, this codon similarly triggers phenotypes in orthogonal assays 56 , 67 , 68 . Hence, the observed effects likely indicate a true biological feature.…”

The translation inhibitor cycloheximide affects ribosome profiling data in a species-specific manner

“…Naturally, prudence will be required when CGA and CGG codons are concerned. However, this may not be specific to ribosome profiling since these two codons also trigger phenotypes in assays that rely on the use of reporter constructs (59)(60)(61)(62). By expressing constructs that contain such rare codons the scarcity of tRNA required for their decoding will be increased.…”