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

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.

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 tRNA ICG 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.
The CGA codon is one of six arginine-encoding codons, as shown in the standard genetic code. In Saccharomyces cerevisiae, CGA is a rare codon decoded by arginyl-tRNA ICG , which is biosynthesized by deamination of the wobble position adenine of tRNA ACG [1]. Arginine codon repeats, which are composed solely or partly of CGA rare codons, have been used for reporter gene constructs, hereafter referred to as 'CGA reporters', to assess the effects of ribosome stalling on mRNAs or translation inhibition [2][3][4][5][6][7]. However, few studies have focused on the decoding aspect [2,4]. Recent ribosome profiling analyses in S. cerevisiae revealed that the use of translation inhibitors, including cycloheximide, to halt protein synthesis resulted in a differential profile at the CGA codon [8], suggesting that CGA decoding in S. cerevisiae could have certain distinctive physiological underpinnings.
Quality control systems for mRNA and nascent polypeptides have been reported in eukaryotic protein synthesis [9][10][11]. Aberrant mRNAs with premature stop codons are degraded via the nonsense mediated mRNA decay (NMD) pathway, with widely conserved UPF factors, and translation termination factors eRF1/eRF3 [12,13]. Aberrant mRNAs stalling ribosomes via formation of stem-loop secondary structures are targeted for degradation via the no-go decay pathway, which includes translation termination factor homologues, Pelota (Dom34 in S. cerevisiae) and Hbs1, on the ribosome [14,15]. Aberrant mRNAs lacking a stop codon are also targeted for degradation via the nonstop decay pathway, wherein in addition to Pelota and HBS1, specific mRNA decay factors, Ski7, the ski complex (ski2/ski3/ski8) and the exosome complex function in mRNA decay [16][17][18]. Ribosomal arrest caused by polybasic residues (such as poly-lysine) or others were reported to cause mRNA cleavage; moreover, nascent polypeptides are degraded via ribosome-associated quality control (RQC) mechanisms [19]. Intriguingly, in the previous analyses, CGA reporters accumulate premature polypeptides (or arrest products) approximately corresponding to the size of the peptide up to the CGA position [2,4,5,20]. Upon knockout of Zuo1, Asc1 affects frame maintenance, which contributes to premature polypeptide synthesis [19,21]. Among those studies on RQC defective conditions including ΔLtn1 or ΔRqcs, CGA reporters yielded premature products with additional mRNA-independent alanine-threonine tails (CATtail) at the C-terminal of nascent-peptides [6,22]. In addition, recent reports on RQC revealed that responses to ribosome arrest or stalling are not homogeneous, depending on the species (S. cerevisiae and mammalian cell line) [20,23,24]. In any eukaryotic processes, ribosomes are supposed to be rescued without polypeptidechain release from P-site peptidyl-tRNA [10,25]. However, in bacteria, three types of ribosome rescue systems are known to function upon ribosomal stalling. tmRNA/ SmpB, ArfA/RF2 and ArfB function in three pathways. Interestingly, ArfB has homologous domains including the GGQ motif to class-I peptide-chain release factors, RF1 and RF2; thus, all three pathways rely on RF or RF-like peptidyl-tRNA hydrolysis activity to release polypeptides from stalled ribosomes [26][27][28]. eRF1 (Sup45 in S. cerevisiae) is a eukaryotic class I polypeptide-chain release factor that directly deciphers UAA, UAG and UGA stop codons in the ribosomal A-site and catalyses the polypeptide chain releasing reaction. Similar to the EF1A/tRNA complex in translation elongation, eRF1 functions in complex with eRF3 (Sup35 in S. cerevisiae) and is referred to as tRNA mimicry [29][30][31][32]. Cooperative interaction between eRF1 and eRF3 is crucial for precise stop codon recognition and translation termination in eukaryotes [32][33][34][35][36][37]. GTPase activity of eRF3 is both eRF1-and ribosome-dependent [33], and their precise interactions on the ribosome have been reported in recent structural analyses [15,31,[38][39][40]. eRF3 is composed of an N-terminal regulatory region and a Cterminal GTPase catalytic region (eRF3c), which is essential for translation termination. The eRF3 Nterminal region interacts with various factors [41,42], and contains unique motif sequences that can induce a [PSI+] prion-like phenotypes [43]. Unlike eukaryotes, in prokaryotes, class I release factors, RF1 and RF2, decipher UAG/UAA and UGA/UAA codons respectively [32,44]. To understand the omnipotency of eRF1, its codon recognition mechanism has been extensively studied [45][46][47][48][49][50]. Well-conserved motifs, known as TASNICS and Y-C-F, are involved in codon recognition at the tip of the codon recognition domain. An eRF1 residue, L123, inside the Y 122 -C 124 -F 128 motif (S. cerevisiae numbering) plays a distinctive role in stop codon discrimination by directly interacting with the ribosome [35,50,51].
Herein, we hypothesized that the CGA codon can be recognized as a stop codon to a certain extent by eRF1/eRF3 translation termination complex in S. cerevisiae, resulting premature polypeptide release and rescue for stalling ribosome on mRNA. We conducted a selection of eRF1 mutants with enhanced termination at CGA codons, and further showed that the wild-type eRF1/eRF3 complex can cause peptide release at the CGA codon in vivo. The putative physiological underpinnings of this phenomenon are also discussed herein.

Results
Isolation of an eRF1 mutant enhancing the viability of the conditional lethal CGA ribosomal stall strain To assess the effect of eRF1 on CGA codon repeats based on growth, we generated a reporter strain. Consecutive 12 CGA codon repeats were cloned immediately upstream to the stop codon of HIS3, and this reporter was introduced into the S. cerevisiae genome, exchanging the HO region to produce the CGA ribosomal stall conditional lethal strain, Y240 (Fig. 1A,B), thus leaving the endogenous wild-type eRF1 and eRF3 chromosomal genes intact. This strain exhibited slight growth on histidine-lacking (-His) media because the consecutive CGA repeats downregulated reporter HIS3 protein (Fig. 1C, 'Vector'). Therefore, growth on histidine-lacking media can be considered to assess the expression of active HIS3 polypeptides, those with CGA codon repeat-mediated prematurely terminated translation and full-length HIS3-R 12 products. To examine the hypothesis that peptide release factor, eRF1, decodes the CGA codon as a stop codon, eRF1 mutants were screened for the enhanced phenotype.  vector, p415GPD (LEU2 selection marker), was randomly mutated via hydroxylamine treatment, introduced into the reporter strain, and viable colonies on SC -His-Leu plates were selected. From approximately 1 9 10 4 eRF1 clones, one most prominently fast-growing clone was specified, and sequencing revealed that the eRF1 mutation was a leucine to phenylalanine substitution (G 369 to C) at position 123   Fig. 1C L123 mutants, F). We decided to focus on the mutant, since eRF1 mutations at this position were extensively analysed [35,51]. Previously, eRF1 position 123 reportedly discriminated the second base of stop codons in an eRF3dependent manner. In the previous study by Saito et al. [35], the eRF1 L123 mutations showed either 'UAA and UAG dual-biased', 'UGA biased' or omnipotent 'UAA, UAG and UGA' specificity similar to wild-type eRF1, via relative read-through efficiency studied in conditional eRF1ts mutant strains. To examine correlations between CGA assay strain growths and stop codon specificities of eRF1 L123 mutants, a set of eRF1 L123 variants (wild-type, F, R, Y, I, V, C and A) was introduced into the reporter strain, and growth was monitored on -His plates (Fig. 1C). The UGA-biased eRF1 mutant transformants, L123F, L123R and L123Y showed marked growth, indicating stable HIS3 expression. eRF1 mutant transformants, L123A and L123C, exhibited growth similar to that of wild-type eRF1. The UAAand UAG-biased eRF1 mutant transformants, L123I and L123V, showed markedly lesser growth than the vector only control and wild-type eRF1 (Fig. 1C). Most growth tendencies shown by the L123 mutants were consistent with eRF1-mediated relative termination activity at the UGA stop codon. Notably, the HIS3-CGA12 reporter gene synthesized sufficient levels of functional HIS3 polypeptide, which is supposed to be released from the stalled ribosome upon expression of mutant eRF1.
To avoid possibility of marked mRNA decay by cellular surveillance, His3 mRNA levels of each transformant were examined via northern blot analysis and quantitative PCR analysis. No significant differences in mRNA levels in eRF1 mutants were observed in our reporter strain (Fig. 1D,E), indicating that the growth of the transformants depends on the efficiency of cellular protein synthesis. These results suggest that eRF1 can potentially recognize CGA codons as stop codons, resulting in the release of premature products.
CGA termination activity of mutant eRF1 assessed via the dual-luciferase assay To further investigate the CGA decoding efficiency of eRF1, quantitative assay strains were generated. Assay strains with dual-luciferase reporter gene constructs, with 12 or 6 CGA codon repeats between Renilla luciferase (Rluc) and luciferase2 (luc2) are hereafter referred to as CGA12-luc reporter and CGA6-luc reporter respectively. The ratio of luc2/Rluc to that of blank assay strain ( Fig. 2) provided an index of the relative levels of the two different sized products, named 'CGA read-through index'. The smaller index putatively shows efficient peptide release at CGA codon repeats. Overexpression of WT eRF1 does not change the CGA read-through index. Using the CGA12-luc reporter, L123F, L123R and L123Y decreased the CGA read-through index, suggesting enhanced peptide release at CGA codon repeats ( Fig. 2A). In contrast, the CGA read-through index was increased by the L123I and L123V mutants. L123A and L123C did not appear to affect CGA read-through. Together, these results indicate that the CGA read-through profile of eRF1 mutants is consistent with the reported 'UGA-specific' read-through profile of eRF1 [21]. Although endogenous eRF1 alleles between previous and the present assay systems are different, the CGA read-through activity is greatly correlated with the UGA read-through activities of the eRF1 variants. This strongly suggests that eRF1 might produce short-sized products from CGA codon repeats by terminating within the CGA codon repeats. Similar CGA read-through index profiles were also observed for eRF1 mutants in the CGA6-luc reporter strain (Fig. 2B). However, read-through index was far higher for the CGA6-luc reporter than for the CGA12-luc reporter. These results indicate that in CGA-luc reporter assays, CGA read-through index ratios are probably the sum of probable peptide release at the CGA codon and reductions in fulllength protein by CGA ribosomal stalling, not a simple reflection of eRF1-mediated termination at CGA codon repeats. Wild-type and mutant eRF1 affects the cellular levels of premature termination products from CGA reporters To directly monitor the cellular levels of premature and full-length products from CGA6-luc and CGA12luc reporters in cells (Fig. 3A), western blot analyses were conducted with the anti-Renilla luciferase (anti-Rluc, aRluc) antibody (Fig. 3B). Since the Rluc ORF is before the CGA codon repeats in the reporter construct, the anti-Rluc antibody should react with fulllength and short-sized (premature) products. As size controls, Rluc-stop (UAA; corresponding to the size of Rluc) and Rluc-R (AGA) 12 -stop (corresponding to the size of Rluc-R 12 ) constructs were included (Fig. 3A). The blot was probed with anti-PGK1 antibody as a loading control (Fig. 3B, lower). In both CGA6-luc and CGA12-luc reporter strains, Rluc-size premature products gradually increased in the order of the vector, wild-type (WT), L123F and L123R (Fig. 3B). Even upon only vector addition in CGA repeat strains, a small amount of short-sized Rluc bands were observed (It is used as basis for the bands ratio). It could be derived from degradation of larger bands, however, even a wild-type effect is evident by quantification. As all strains used have endogenous eRF1 and eRF3, those probably contribute to the production of short-sized Rluc bands at the CGA repeats.
These results confirmed that the short-sized products are Rluc released by eRF1. There was no difference in the sizes of premature products produced from CGA6-luc and CGA12-luc reporter strains. From the comparison with Rluc and Rluc-R 12 size markers, surprisingly, only one, or a few, CGA codon(s) seem to be required to cause peptide release in S. cerevisiae, although many CGA codons are required to investigate this effect in reporter systems. Although similar size bands were observed in blank strain transformants, they are independent of the CGA presence and insensitive to eRF1 overproduction. Full-length product levels were not prominently reduced upon an increase in the short-sized products in the CGA6-luc reporter strain (Fig. 3B CGA6-luc), although the amount of the full-length product is much lesser than those of the blank strain (Fig. 3B Blank). Full-length products were only faintly observed in the CGA12-luc reporter strain probably owing to a pleiotropic effect by accumulated translation inefficiency through a much longer CGA repeat.
Northern blot analysis revealed that the mRNA levels detected by Rluc probe in each sample are comparable (Fig. 3C), indicating that CGA6-luc and CGA12-luc reporter mRNAs are not subjected to significant intracellular mRNA decay. To examine the competition between eRF1 and tRNAs in the CGA6-luc reporter strain, tRNA ACG and tRNA UCG expressing plasmids were constructed. Since CGA arginine codon is deciphered by tRNA ICG from tRNA ACG via post-transcriptional modification in S. cerevisiae, tRNA ACG gene encoding tRNA ICG precursor that is believed to be less efficient for decoding, and mutant tRNA UCG , which is mutated from tRNA ACG to perfectly match to decipher CGA without cellular modification, were cloned into pRS416 (single copy plasmid).
Either of vector (p415GPD), WT, L123F and L123R eRF1 in p415GPD was co-introduced with either of indicated tRNA-encoding plasmids into the reporter strain and products were detected via western blot analysis (Fig. 4A). Consequently, reduction in the short-sized premature products (Rluc*) and reciprocal increase in the full-length products were observed with tRNA UCG in all of vector, WT, L123F and L123R eRF1 expressing strains. The effect was not evident with tRNA ACG . The result seems ambiguous from the point of competition with eRF1 and tRNA ICG , that should exist a lot in vivo, in this experiment. However, instead, it shows rather strong effect of eRF1 at CGA.
Homogeneous eRF3 cooperatively enhances eRF1-mediated CGA decoding To compare the manner of recognition of the CGA codon by the eRF1/eRF3 translation termination complex with the canonical translation termination at (Hs)) were overexpressed in the CGA6-luc reporter strain and assessed via western blot analysis (Fig. 4B). Co-overproduction of homogeneous yeast eRF1 and eRF3 enhanced the production of short-sized premature polypeptides (Fig. 4B, WT). This effect was more prominent upon expression of the L123F eRF1 mutant. On the contrary, heterogeneous expression of human eRF3 (GSPT1c) with yeast eRF1 hardly enhanced the production of short-sized premature polypeptides, despite GSPT1c's replaceability the endogenous activity of yeast eRF3. The present results show that the homogeneous combination of yeast eRF1/eRF3 recognizes the CGA codon in a manner similar to that of stop codons during translation termination.
A single CGA codon at the end of the gene is detectably decoded as the stop codon The present results indicate that only a few CGA codons are sufficient for the eRF1/eRF3 complex to cause peptide release within the repeats in a competitive manner with 'inefficient' decoding of CGA by the tRNA/EF1A complex. Therefore, we investigated the effect of a single CGA codon under a condition favourable for the eRF1/eRF3 complex, that is at a position for normal stop codons within the mRNA (Fig. 5). The Rluc-TGA-his3t (HIS3 3 0 UTR and terminator) plasmid reporter was constructed, and the TGA stop codon of the reporter was exchanged for CGA, and AGA, GGA and AGG control codons (Fig. 5A). The BY4727 wild-type strain was transformed with reporter plasmids and Rluc expression was examined via western blot analysis. Using the Rluc-CGA-his3t reporter, we observed slight but significant synthesis of the Rluc product, whereas no Rluc product was observed in transformants expressing reporters with AGA, GGA and AGG codons (Fig. 5B left). Overexpression of eRF1 L123R mutant with the Rluc-CGA reporter revealed enhanced Rluc products (Fig. 5B  right). These data provide strong evidence that the endogenous eRF1/eRF3 translation termination complex can recognize CGA to release polypeptides. Although signal enhancement by WT eRF1 overproduction is not observed in the system, it probably reflects unknown requirements for peptide release at single CGA, showing clear difference in efficiency with proper translation termination in the context.

Discussion
In this study, several artificial reporter systems were introduced to confirm that the eRF1/eRF3 complex can cause premature peptide release at CGA sense codons in S. cerevisiae. A specific sense codon, CGA, is decoded as a stop codon by the translation termination complex, eRF1/eRF3. The biological underpinnings of its latent nature are yet unclear. However, recent ribosome profiling data exhibited potential slow decoding of CGA codons on various mRNAs, suggesting their regulatory significance together with its low codon usage [8,52].  the capacity to read CGA codons. As eRF3 has Nterminal regulatory region that interacts with plural factors [41,54], direct and indirect interactions with the factors or mRNA may affect, as it were, 'CGA recoding', in addition to tRNA ICG availability [55,56]. Recent reports evidenced the dual use of stop codons as 'stop' and 'sense' codons in some organisms, depending on their location [57][58][59][60]. CGA codon deciphered via translation termination factors, the eRF1/eRF3 protein complex, may be an evolutionary deviation demonstrating a flexible eukaryotic translation termination system.
From the viewpoint of quality control, CGA repeats could provide a distinctive mode of ribosome stalling among other ribosome stall reporters, as shown via mRNA quantitation analyses performed herein (Figs 1D,E and 3C) and previously [2,5,20]. On performing western blot analyses in this study, the CGAluc reporters yielded two distinctive protein products, that is short-sized and full-length ones. The amount of the full-length product contrasts the number of CGA, as is previously reported with reporter constructs [2,20] and seemingly unaffected by eRF activities. However, intriguingly, the amount of the short-sized product is seemingly unaffected by the number of CGA, while being susceptible to the putative CGA misdecoding activity of wild-type eRF1 and to that of L123 mutated eRF1 in a cooperative manner with eRF3. We believe that short-sized product seems to be released from the stalled ribosomes by decoding of early CGA within consecutive CGAs, whereas fulllength product synthesis is inhibited depending on the number of CGAs. The precise mechanism underlying the reduction of the full-length product at the CGA repeats is yet unknown; however, we hypothesize that inefficient decoding by tRNA ICG is involved and it also enables easy visualization of rather inefficient short-sized band production in the system. Our hypothesis is not inconsistent with the latest ribosome profiling analysis of the stalled ribosomes on poly-Arg codons containing CGAs [20].
Recent advancements in cryo-EM revealed structural aspects of stop codon decoding by eRF1 in the ribosome [38,39,[61][62][63]. In the pre-GTP hydrolysis mode [38] and in the accommodation mode [61], conformation of domain1 of eRF1 and A-site stop codon appears similar (Fig. 6), suggesting that domain1 conformation is not largely affected during this process. Based on those latest structures, the trinucleotides region of the stop codon, surprisingly, form U-turns, and the structure seems to be primarily fixed by interactions between NIKS 58-61 motif (Hs (Human numbering):NIKS [61][62][63][64] ) and 1st base U, 3rd nucleotide backbone, between A1825 (A2256 in S. cerevisiae, A1493 in Escherichia coli) of 18S rRNA h44 and 2nd base. The NIKS motif interacts with 1st base of the codon, and part of Y 122 -C 124 -F 128 (Hs:Y 125 -C 127 -F 131 )motif and E 52 (Hs:E 55 ) with 2nd and 3rd bases [61]. These interactions coincide with those reported in previous genetic studies [35,47,64,65]. However, in our previous study, we speculated functional counterpart regions between eRF1 and tRNA, by structural superimposition of EF-Tu/tRNA complex and archeal translation termination aEF1A/aRF1 complex based on tRNA mimicry [34]. In this prediction, L123 residue (R126 in A. pernix) corresponds to the backbone moieties around the 38th and 39th nucleotides of tRNA in close proximity of the 36th base of tRNA complementary to the 1st base of A-site codon, possibly affecting decoding fidelity. The protruding side chain moiety of L123 are proximal to and potentially interact with both 25S rRNA (A3731 of helix69, Hs numbering) and 18S rRNA (A1825 that makes stack with 2nd base of stop codon), whereas the side chains of adjacent residues Y 122 and C 124 is directed to the opposite direction to form stop codon binding pocket in the ribosomal complex structures. E 52 of eRF1 occupies rather specific position that reported to interact the 2nd and the 3rd bases depending on the stop codons, that is it may contribute to stop codon discrimination directly [61]. The L123 of eRF1 was speculated to play its role through the 2nd base discrimination by Y 122-C 124 and E 52 .
The C4 carbonyl of uracil at the 1st base of stop codon interacts with K60 (Hs:K 63 ) within the NIKS motif. For the putative CGA misdecoding in our hypothesis, an amino group of cytosine at the same position would deter its interaction with K60, without largely loosing other interactions. Although K60 comprises important conserved motifs, in previous reports, single K60 mutant eRF1s did not completely lose their activity towards stop codons, suggesting potential recognition of CGA by eRF1 [49,64]. L123 of eRF1 may be functionally sensitive for the distance between the 1st base and ribosomal interaction subunits, making bulkier mutations (F, R, Y) functionally easier in both stop and CGA codons. Interestingly, in human eRF1, K60 (Hs: K63), which is proximal to the 1st base of the stop codon, within TASNIKS motif was reported to be modified C4 lysyl hydroxylation by JMJD4. However, the modification enzyme was not found in S. cerevisiae; thus, the 1st base decoding might be differently regulated among species. Sense codon misdecoding capacities of eRF1 should be studied in detail in future studies. The physiological Intriguingly, if eRF1/eRF3 complex can rescue stalled ribosomes, producing premature products within inefficiently translated mRNA contexts such like CGA repeats, it is tempting to assume that it could provide an analogous situation as NMD, wherein eRF1/eRF3 releases the polypeptide at the premature termination codon. Recently, NMD was reported to be involved in translational fidelity [13] and physiological regulation [66], but not only for unexpected decay of aberrant mRNA. Therefore, misdecoding by eRF1/eRF3, as in CGA codon repeats, is strongly suggested to have global regulatory underpinnings, which are yet unknown. It is interesting to note that codon usage and the corresponding tRNA repertoire in organisms may be a part of sophisticated translational regulatory systems.

Strains and plasmids
The strains and the plasmids used in this study are listed in Table 1

Mutant isolation and growth assay
The eRF1 gene cloned into p415GPD (pMW1111) was randomly mutated via hydroxylamine treatment. Five micrograms of pMW1111 was added to hydroxylamine solution (1 M hydroxylamine, 0.4 lM EDTA, pH 6.0) and incubated at 37°C for 20 h. Plasmids were ethanol precipitated and washed with 70% ethanol, twice. The treated pMW1111 plasmids were introduced into the HIS3-CGA 12stop growth reporter strain, Y240, via transformation. Y240 transformants were then incubated on SC-His-Leu agar plates at 30°C to select for growing colonies (pMW1106). For growth assay, Y240 strain was transformed with vector or eRF1 variants (pMW1111 and pMW1112s; pMW1112 L123F is equivalent to the screened pMW1106) and incubated on SC-Leu agar plates at 30°C. Obtained transformants were seeded onto SC-His-Leu agar plates and incubated at 30°C to examine growth.

CGA read-through assay
The dual-luciferase translational read-through assay was conducted in accordance with a previously reported method [35]. Y262, Y294 and Y261 (blank) strains were transformed with p415GPD vector or eRF1 variants cloned in p415GPD. The transformants were cultured at 30°C, cells were harvested and suspended in lysis buffer, and cell lysates were prepared by vigorously shaking with glass beads (Sigma-Aldrich, St. Louis, MO, USA), using Fastprep24 (MP biomedicals, Santa Ana, CA, USA). The dual-luciferase assay was conducted with Dual-luciferase Reporter Assay System (Promega, Madison, WI, USA) in accordance with the manufacturer's instructions. The percentage of CGA

Western blot analysis
The transformant cells were cultured in appropriate media till the mid-log phase (OD 600 of approximately 0.75). The cells were harvested, treated with 10% TCA for 30 min on ice and harvested. Thereafter, the samples were suspended in sample buffer (Laemmli, Sigma-Aldrich), and the neutral pH was adjusted with 4 N NaOH and the mixture was vigorously shaken with glass beads (Sigma-Aldrich), using Fastprep24 (MP biomedicals). The protein in the lysates were separated via SDS/PAGE (10% resolving gel) and transferred to Hybond-ECL (GE Healthcare, Little Chalfont, UK) via the semidry method. The filters were treated with either anti-Rluc Antibody, clone 5B11.2 (anti-Rluc; Merck Millipore, Billerica, MA, USA) or PGK1 monoclonal antibody (anti-PGK; 22C5D8; ThermoFisher), and Anti-Mouse IgG, HRP-linked Whole Ab Sheep (GE Healthcare). Signals were detected with ImmunoStar LD (Wako Laboratory Chemicals, Osaka, Japan) and LAS-3000 mini (Fujifilm, Tokyo, Japan). Quantification of bands was conducted by Multi Gauge software (Fujifilm). To detect anti-Rluc antibody, the Can Get Signal Immunoreaction enhancer (Toyobo, Osaka, Japan) was used to enhance the reaction.

Northern blot analysis
The transformant cells were cultured in the appropriate media to the mid-log phase. The cells were harvested and samples were prepared as described previously [67], via hot phenol treatment and ethanol precipitation, and dissolved in H 2 O. In 20 lg of each RNA sample, MOPS buffer with formaldehyde and formamide was added, and incubated at 55°C for 10 min. RNA samples were separated via electrophoresis on a 1.2% agarose gel with 6.7% formaldehyde, then transferred to Hybond-N+ (GE Healthcare) and fixed via UV irradiation. RNAs were detected using the ECL Direct Nucleic Acid Labeling and Detection System (GE Healthcare) in accordance with the manufacturer's protocol. The His3 probe was amplified via PCR from p413GPD, using primers P768/P769 (5 0 -ACGACCATCACACCACTGAA-3 0 / 5 0 -GCGAGGTGGCTTCTCTTATG-3 0 ). The Rluc probe was amplified via PCR from pMW1208 with primers P828/P829 (5 0 -CGAGCACCAAGACAAGATCA -3 0 /5 0 -CATTTCAT CTGGAGCGTCCT -3 0 ), and the SCR1 probe was amplified from pMW1326 with primers P384/P385 (5 0 -AGGCTGTA ATGGCTTTCTGGTGGGATGGGA-3 0 /5 0 -GATATGTGCT ATCCCGGCCGCCTCCATCAC-3 0 ). The probes were labelled and used for hybridization to the filter. The filter was washed with the primary wash buffer (0.5 9 SSC, 0.4% SDS and 6 M urea) and the secondary wash buffer (2 9 SSC), followed by detection with the detection solution and measured using LAS-3000 mini (Fujifilm).