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1.  Overexpression of human release factor 1 alone has an antisuppressor effect in human cells. 
Molecular and Cellular Biology  1997;17(6):3164-3172.
Two eukaryotic proteins involved in translation termination have recently been characterized in in vitro experiments. Eukaryotic release factor 1 (eRF1) catalyzes the release of the polypeptide chain without any stop codon specificity. The GTP-binding protein eRF3 confers GTP dependence to the termination process and stimulates eRF1 activity. We used tRNA-mediated nonsense suppression at different stop codons in a cat reporter gene to analyze the polypeptide chain release factor activities of the human eRF1 and eRF3 proteins overexpressed in human cells. In a chloramphenicol acetyltransferase assay, we measured the competition between the suppressor tRNA and the human release factors when a stop codon was present in the ribosomal A site. Whatever the stop codon (UAA, UAG, or UGA) present in the cat open reading frame, the overexpression of human eRF1 alone markedly decreased translational readthrough by suppressor tRNA. Thus, like the procaryotic release factors RF1 and RF2 in Escherichia coli, eRF1 seems to have an intrinsic antisuppressor activity in human cells. Levels of antisuppression of overexpression of both eRF3 and eRF1 were almost the same as those of overexpression of eRF1 alone, suggesting that eRF1-eRF3 complex-mediated termination may be controlled by the expression level of eRF1. Surprisingly, when overexpressed alone, eRF3 had an inhibitory effect on cat gene expression. The results of cat mRNA stability studies suggest that eRF3 inhibits gene expression at the transcriptional level. This indicates that in vivo, eRF3 may perform other functions, including the stimulation of eRF1 activity.
PMCID: PMC232169  PMID: 9154815
2.  N-terminal region of Saccharomyces cerevisiae eRF3 is essential for the functioning of the eRF1/eRF3 complex beyond translation termination 
Termination of translation in eukaryotes requires two release factors, eRF1, which recognizes all three nonsense codons and facilitates release of the nascent polypeptide chain, and eRF3 stimulating translation termination in a GTP-depended manner. eRF3 from different organisms possess a highly conservative C region (eRF3C), which is responsible for the function in translation termination, and almost always contain the N-terminal extension, which is inessential and vary both in structure and length. In the yeast Saccharomyces cerevisiae the N-terminal region of eRF3 is responsible for conversion of this protein into the aggregated and functionally inactive prion form.
Here, we examined functional importance of the N-terminal region of a non-prion form of yeast eRF3. The screen for mutations which are lethal in combination with the SUP35-C allele encoding eRF3C revealed the sup45 mutations which alter the N-terminal domain of eRF1 and increase nonsense codon readthrough. However, further analysis showed that synthetic lethality was not caused by the increased levels of nonsense codon readthrough. Dominant mutations in SUP35-C were obtained and characterized, which remove its synthetic lethality with the identified sup45 mutations, thus indicating that synthetic lethality was not due to a disruption of interaction with proteins that bind to this eRF3 region.
These and other data demonstrate that the N-terminal region of eRF3 is involved both in modulation of the efficiency of translation termination and functioning of the eRF1/eRF3 complex outside of translation termination.
PMCID: PMC1617110  PMID: 17034622
3.  The effect of eukaryotic release factor depletion on translation termination in human cell lines 
Nucleic Acids Research  2004;32(15):4491-4502.
Two competing events, termination and readthrough (or nonsense suppression), can occur when a stop codon reaches the A-site of a translating ribosome. Translation termination results in hydrolysis of the final peptidyl-tRNA bond and release of the completed nascent polypeptide. Alternatively, readthrough, in which the stop codon is erroneously decoded by a suppressor or near cognate transfer RNA (tRNA), results in translation past the stop codon and production of a protein with a C-terminal extension. The relative frequency of termination versus readthrough is determined by parameters such as the stop codon nucleotide context, the activities of termination factors and the abundance of suppressor tRNAs. Using a sensitive and versatile readthrough assay in conjunction with RNA interference technology, we assessed the effects of depleting eukaryotic releases factors 1 and 3 (eRF1 and eRF3) on the termination reaction in human cell lines. Consistent with the established role of eRF1 in triggering peptidyl-tRNA hydrolysis, we found that depletion of eRF1 enhances readthrough at all three stop codons in 293 cells and HeLa cells. The role of eRF3 in eukarytotic translation termination is less well understood as its overexpression has been shown to have anti-suppressor effects in yeast but not mammalian systems. We found that depletion of eRF3 has little or no effect on readthrough in 293 cells but does increase readthrough at all three stop codons in HeLa cells. These results support a direct role for eRF3 in translation termination in higher eukaryotes and also highlight the potential for differences in the abundance or activity of termination factors to modulate the balance of termination to readthrough reactions in a cell-type-specific manner.
PMCID: PMC516063  PMID: 15326224
4.  Involvement of Human Release Factors eRF3a and eRF3b in Translation Termination and Regulation of the Termination Complex Formation 
Molecular and Cellular Biology  2005;25(14):5801-5811.
eRF3 is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. Both bind eRF1 and stimulate its release activity in vitro. However, whether both proteins can function as termination factors in vivo has not been determined. In this study, we used short interfering RNAs to examine the effect of eRF3a and eRF3b depletion on translation termination efficiency in human cells. By measuring the readthrough at a premature nonsense codon in a reporter mRNA, we found that eRF3a silencing induced an important increase in readthrough whereas eRF3b silencing had no significant effect. We also found that eRF3a depletion reduced the intracellular level of eRF1 protein by affecting its stability. In addition, we showed that eRF3b overexpression alleviated the effect of eRF3a silencing on readthrough and on eRF1 cellular levels. These results suggest that eRF3a is the major factor acting in translation termination in mammals and clearly demonstrate that eRF3b can substitute for eRF3a in this function. Finally, our data indicate that the expression level of eRF3a controls the formation of the termination complex by modulating eRF1 protein stability.
PMCID: PMC1168810  PMID: 15987998
5.  Elongation factor eEF1B modulates functions of the release factors eRF1 and eRF3 and the efficiency of translation termination in yeast 
BMC Molecular Biology  2009;10:60.
Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. While eRF1 recognizes nonsense codons, eRF3 facilitates polypeptide chain release from the ribosome in a GTP-dependent manner. Besides termination, both release factors have essential, but poorly characterized functions outside of translation.
To characterize further the functions of yeast eRF1 and eRF3, a genetic screen for their novel partner proteins was performed. As a result, the genes for γ (TEF4 and TEF3/CAM1) and α (TEF5/EFB1) subunits of the translation elongation factor eEF1B, known to catalyze the exchange of bound GDP for GTP on eEF1A, were revealed. These genes act as dosage suppressors of a synthetic growth defect caused by some mutations in the SUP45 and SUP35 genes encoding eRF1 and eRF3, respectively. Extra copies of TEF5 and TEF3 can also suppress the temperature sensitivity of some sup45 and sup35 mutants and reduce nonsense codon readthrough caused by these omnipotent suppressors. Besides, overproduction of eEF1Bα reduces nonsense codon readthrough in the strain carrying suppressor tRNA. Such effects were not shown for extra copies of TEF2, which encodes eEF1A, thus indicating that they were not due to eEF1A activation.
The data obtained demonstrate involvement of the translation elongation factor eEF1B in modulating the functions of translation termination factors and suggest its possible role in GDP for GTP exchange on eRF3.
PMCID: PMC2705663  PMID: 19545407
6.  Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA. 
Nucleic Acids Research  1997;25(12):2254-2258.
It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.
PMCID: PMC146740  PMID: 9171074
7.  GTP Hydrolysis by eRF3 Facilitates Stop Codon Decoding during Eukaryotic Translation Termination 
Molecular and Cellular Biology  2004;24(17):7769-7778.
Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.
PMCID: PMC506980  PMID: 15314182
8.  Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components 
Members of the eukaryote/archaea specific eRF1 and eRF3 protein families have central roles in translation termination. They are also central to various mRNA surveillance mechanisms, together with the eRF1 paralogue Dom34p and the eRF3 paralogues Hbs1p and Ski7p. We have examined the evolution of eRF1 and eRF3 families using sequence similarity searching, multiple sequence alignment and phylogenetic analysis.
Extensive BLAST searches confirm that Hbs1p and eRF3 are limited to eukaryotes, while Dom34p and eRF1 (a/eRF1) are universal in eukaryotes and archaea. Ski7p appears to be restricted to a subset of Saccharomyces species. Alignments show that Dom34p does not possess the characteristic class-1 RF minidomains GGQ, NIKS and YXCXXXF, in line with recent crystallographic analysis of Dom34p. Phylogenetic trees of the protein families allow us to reconstruct the evolution of mRNA surveillance mechanisms mediated by these proteins in eukaryotes and archaea.
We propose that the last common ancestor of eukaryotes and archaea possessed Dom34p-mediated no-go decay (NGD). This ancestral Dom34p may or may not have required a trGTPase, mostly like a/eEF1A, for its delivery to the ribosome. At an early stage in eukaryotic evolution, eEF1A was duplicated, giving rise to eRF3, which was recruited for translation termination, interacting with eRF1. eRF3 evolved nonsense-mediated decay (NMD) activity either before or after it was again duplicated, giving rise to Hbs1p, which we propose was recruited to assist eDom34p in eukaryotic NGD. Finally, a third duplication within ascomycete yeast gave rise to Ski7p, which may have become specialised for a subset of existing Hbs1p functions in non-stop decay (NSD). We suggest Ski7p-mediated NSD may be a specialised mechanism for counteracting the effects of increased stop codon read-through caused by prion-domain [PSI+] mediated eRF3 precipitation.
PMCID: PMC2613156  PMID: 18947425
9.  Viable nonsense mutants for the essential gene SUP45 of Saccharomyces cerevisiae 
Termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) – eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45.
We have isolated five sup45-n (n from nonsense) mutations that cause nonsense substitutions in the following amino acid positions of eRF1: Y53 → UAA, E266 → UAA, L283 → UAA, L317 → UGA, E385 → UAA. We found that full-length eRF1 protein is present in all mutants, although in decreased amounts. All mutations are situated in a weak termination context. All these sup45-n mutations are viable in different genetic backgrounds, however their viability increases after growth in the absence of wild-type allele. Any of sup45-n mutations result in temperature sensitivity (37°C). Most of the sup45-n mutations lead to decreased spore viability and spores bearing sup45-n mutations are characterized by limited budding after germination leading to formation of microcolonies of 4–20 cells.
Nonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.
PMCID: PMC150568  PMID: 12589713
10.  A single amino acid change of translation termination factor eRF1 switches between bipotent and omnipotent stop-codon specificity† 
Nucleic Acids Research  2010;39(2):599-608.
In eukaryotes a single class-1 translation termination factor eRF1 decodes the three stop codons: UAA, UAG and UGA. Some ciliates, like Euplotes, have a variant code, and here eRF1s exhibit UAR-only specificity, whereas UGA is reassigned as a sense codon. Since eukaryote eRF1 stop-codon recognition is associated with its N-terminal domain, structural features should exist in the N domain of ciliate eRF1s that restrict their stop-codon specificity. Using an in vitro reconstituted eukaryotic translation system we demonstrate here that a chimeric eRF1 composed of the N domain of Euplotes aediculatus eRF1 fused to the MC domains of human eRF1 exhibits UAR-only specificity. Functional analysis of eRF1 chimeras constructed by swapping Euplotes N domain sequences with the cognate regions from human eRF1 as well as site-directed mutagenesis of human eRF1 highlighted the crucial role of the alanine residue in position 70 of E. aediculatus eRF1 in restricting UGA decoding. Switching the UAR-only specificity of E. aediculatus eRF1 to omnipotent mode is due to a single point mutation. Furthermore, we examined the influence of eRF3 on the ability of chimeric and mutant eRF1s to induce peptide release in response to different stop codons.
PMCID: PMC3025575  PMID: 20860996
11.  GTP-dependent structural rearrangement of the eRF1:eRF3 complex and eRF3 sequence motifs essential for PABP binding 
Nucleic Acids Research  2009;38(2):548-558.
Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation.
PMCID: PMC2811017  PMID: 19906736
12.  Termination of translation in eukaryotes is mediated by the quaternary eRF1•eRF3•GTP•Mg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct 
Nucleic Acids Research  2006;34(14):3947-3954.
GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg2+. We show that (i) eRF3 binds GDP (Kd = 1.9 μM) and this interaction depends only minimally on the Mg2+ concentration; (ii) GTP binds to eRF3 (Kd = 0.5 μM) only in the presence of eRF1 and this interaction depends on the Mg2+ concentration; (iii) GTP displaces GDP from the eRF1•eRF3•GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1•eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1•eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1•eRF3•GTP•Mg2+, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3•eRF1 complex profoundly differ from those of prokaryotic RF3.
PMCID: PMC1557817  PMID: 16914449
13.  Mutation at Tyrosine in AMLRY (GILRY Like) Motif of Yeast eRF1 on Nonsense Codons Suppression and Binding Affinity to eRF3 
Termination translation in Saccharomyces cerevisiae is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. Two regions in human eRF1, position at 281-305 and position at 411-415, were proposed to be involved on the interaction to eRF3. In this study we have constructed and characterized yeast eRF1 mutant at position 410 (correspond to 415 human eRF1) from tyrosine to serine residue resulting eRF1(Y410S). The mutations did not affect the viability and temperature sensitivity of the cell. The stop codons suppression of the mutant was analyzed in vivo using PGK-stop codon-LACZ gene fusion and showed that the suppression of the mutant was significantly increased in all of codon terminations. The suppression on UAG codon was the highest increased among the stop codons by comparing the suppression of the wild type respectively. In vitro interaction between eRF1 (mutant and wild type) to eRF3 were carried out using eRF1-(His)6 and eRF1(Y410S)-(His)6 expressed in Escherichia coli and indigenous Saccharomyces cerevisiae eRF3. The results showed that the binding affinity of eRF1(Y410S) to eRF3 was decreased up to 20% of the wild type binding affinity. Computer modeling analysis using Swiss-Prot and Amber version 9.0 programs revealed that the overall structure of eRF1(Y410S) has no significant different with the wild type. However, substitution of tyrosine to serine triggered the structural change on the other motif of C-terminal domain of eRF1. The data suggested that increasing stop codon suppression and decreasing of the binding affinity of eRF1(Y410S) were probably due to the slight modification on the structure of the C-terminal domain.
PMCID: PMC2359899  PMID: 18463713
eRF1; eRF3; Saccharomyces cerevisiae; termination translation; nonsense codon suppression; binding affinity
14.  Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus 
Journal of Virology  2014;88(18):10364-10376.
Translational readthrough—suppression of termination at a stop codon—is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an ∼8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial.
IMPORTANCE Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational “recoding” processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.
PMCID: PMC4178896  PMID: 24991001
15.  Distinct eRF3 Requirements Suggest Alternate eRF1 Conformations Mediate Peptide Release During Eukaryotic Translation Termination 
Molecular cell  2008;30(5):599-609.
Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, while variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.
PMCID: PMC2475577  PMID: 18538658
16.  Distinct Paths To Stop Codon Reassignment by the Variant-Code Organisms Tetrahymena and Euplotes 
Molecular and Cellular Biology  2006;26(2):438-447.
The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed that changes in domain 1 of ciliate eRF1s are responsible for altered stop codon recognition, this has never been demonstrated in vivo. To carry out such an analysis, we made hybrid proteins that contained eRF1 domain 1 from either Tetrahymena thermophila or Euplotes octocarinatus fused to eRF1 domains 2 and 3 from Saccharomyces cerevisiae. We found that the Tetrahymena hybrid eRF1 efficiently terminated at all three stop codons when expressed in yeast cells, indicating that domain 1 is not the sole determinant of stop codon recognition in Tetrahymena species. In contrast, the Euplotes hybrid facilitated efficient translation termination at UAA and UAG codons but not at the UGA codon. Together, these results indicate that while domain 1 facilitates stop codon recognition, other factors can influence this process. Our findings also indicate that these two ciliate species used distinct approaches to diverge from the universal genetic code.
PMCID: PMC1346903  PMID: 16382136
17.  Readthrough of nonsense mutation W822X in the SCN5A gene can effectively restore expression of cardiac Na+ channels 
Cardiovascular Research  2009;83(3):473-480.
Nonsense mutations in the SCN5A gene result in truncated, non-functional derivatives of the cardiac Na+ channel and thus cause arrhythmias. Studies of other genes suggest that pathogenic phenotypes of nonsense mutations may be alleviated by enhancing readthrough, which enables ribosomes to ignore premature termination codons and produce full-length proteins. Thus, we studied the functional restoration of nonsense-mutated SCN5A.
Methods and results
HEK293 cells were transfected with SCN5A cDNA or its mutant carrying W822X, a nonsense mutation associated with Brugada syndrome and sudden cardiac death. The effects of readthrough-enhancing reagents on Na+ channel expression and function were examined in the transfected cells. W822X robustly reduced Na+ current, decreasing maximal Na+ current to <3% of the wild-type level, and inhibited the expression of full-length Na+ channels. When readthrough was enhanced by either reducing translational fidelity with aminoglycosides or decreasing translation termination efficiency with small-interfering RNA against eukaryotic release factor eRF3a, Na+ current of the mutant was restored to ∼30% of the wild-type level; western blot and immunochemical staining analyses showed the increased expression of full-length channels. When the wild-type and mutant cDNAs were co-transfected, readthrough-enhancing reagents increased Na+ current from 56% to 74% of the wild-type level. Analysis of Na+ channel kinetics showed that the channels expressed from the mutant cDNA under readthrough-enhancing conditions retained the functions of wild-type channels.
Readthrough-enhancing reagents can effectively suppress SCN5A nonsense mutations and may restore the expression of full-length Na+ channels with normal functions, which might prevent sudden cardiac death in mutation carriers.
PMCID: PMC2709462  PMID: 19377070
Sodium channels; Aminoglycosides; Eukaryotic release factors; siRNA; Nonsense mutation
18.  Optimal Translational Termination Requires C4 Lysyl Hydroxylation of eRF1 
Molecular Cell  2014;53(4):645-654.
Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation.
Graphical Abstract
•Jmjd4 hydroxylates translational termination factor eRF1•The C4 lysyl hydroxylase activity of Jmjd4 is unprecedented in animals•Hydroxylation occurs within the eRF1 stop codon recognition domain•Inhibiting eRF1 K63 hydroxylation promotes stop codon readthrough
Accurate termination of protein synthesis at a stop codon is essential in order to maintain protein sequence fidelity. Feng et al. show that translational termination is optimized by lysyl hydroxylation of the eukaryotic release factor eRF1 by Jmjd4, a 2-oxoglutarate- and Fe(II)-dependent oxygenase.
PMCID: PMC3991326  PMID: 24486019
19.  A novel mutant of the Sup35 protein of Saccharomyces cerevisiae defective in translation termination and in GTPase activity still supports cell viability 
When a stop codon is located in the ribosomal A-site, the termination complex promotes release of the polypeptide and dissociation of the 80S ribosome. In eukaryotes two proteins eRF1 and eRF3 play a crucial function in the termination process. The essential GTPase Sup35p, the eRF3 release factor of Saccharomyces cerevisiae is highly conserved. In particular, we observed that all eRF3 homologs share a potential phosphorylation site at threonine 341, suggesting a functional role for this residue. The goal of this study was to determine whether this residue is actually phosphorylated in yeast and if it is involved in the termination activity of the protein.
We detected no phosphorylation of the Sup35 protein in vivo. However, we show that it is phosphorylated by the cAMP-dependent protein kinase A on T341 in vitro. T341 was mutated to either alanine or to aspartic acid to assess the role of this residue in the activity of the protein. Both mutant proteins showed a large decrease of GTPase activity and a reduced interaction with eRF1/Sup45p. This was correlated with an increase of translational readthrough in cells carrying the mutant alleles. We also show that this residue is involved in functional interaction between the N- and C-domains of the protein.
Our results point to a new critical residue involved in the translation termination activity of Sup35 and in functional interaction between the N- and C-domains of the protein. They also raise interesting questions about the relation between GTPase activity of Sup35 and its essential function in yeast.
PMCID: PMC2259375  PMID: 18267004
20.  Decoding accuracy in eRF1 mutants and its correlation with pleiotropic quantitative traits in yeast 
Nucleic Acids Research  2010;38(16):5479-5492.
Translation termination in eukaryotes typically requires the decoding of one of three stop codons UAA, UAG or UGA by the eukaryotic release factor eRF1. The molecular mechanisms that allow eRF1 to decode either A or G in the second nucleotide, but to exclude UGG as a stop codon, are currently not well understood. Several models of stop codon recognition have been developed on the basis of evidence from mutagenesis studies, as well as studies on the evolutionary sequence conservation of eRF1. We show here that point mutants of Saccharomyces cerevisiae eRF1 display significant variability in their stop codon read-through phenotypes depending on the background genotype of the strain used, and that evolutionary conservation of amino acids in eRF1 is only a poor indicator of the functional importance of individual residues in translation termination. We further show that many phenotypes associated with eRF1 mutants are quantitatively unlinked with translation termination defects, suggesting that the evolutionary history of eRF1 was shaped by a complex set of molecular functions in addition to translation termination. We reassess current models of stop-codon recognition by eRF1 in the light of these new data.
PMCID: PMC2938225  PMID: 20444877
21.  Eukaryotic Release Factor 1 Phosphorylation by CK2 Protein Kinase Is Dynamic but Has Little Effect on the Efficiency of Translation Termination in Saccharomyces cerevisiae 
Eukaryotic Cell  2006;5(8):1378-1387.
Protein synthesis requires a large commitment of cellular resources and is highly regulated. Previous studies have shown that a number of factors that mediate the initiation and elongation steps of translation are regulated by phosphorylation. In this report, we show that a factor involved in the termination step of protein synthesis is also subject to phosphorylation. Our results indicate that eukaryotic release factor 1 (eRF1) is phosphorylated in vivo at serine 421 and serine 432 by the CK2 protein kinase (previously casein kinase II) in the budding yeast Saccharomyces cerevisiae. Phosphorylation of eRF1 has little effect on the efficiency of stop codon recognition or nonsense-mediated mRNA decay. Also, phosphorylation is not required for eRF1 binding to the other translation termination factor, eRF3. In addition, we provide evidence that the putative phosphatase Sal6p does not dephosphorylate eRF1 and that the state of eRF1 phosphorylation does not influence the allosuppressor phenotype associated with a sal6Δ mutation. Finally, we show that phosphorylation of eRF1 is a dynamic process that is dependent upon carbon source availability. Since many other proteins involved in protein synthesis have a CK2 protein kinase motif near their extreme C termini, we propose that this represents a common regulatory mechanism that is shared by factors involved in all three stages of protein synthesis.
PMCID: PMC1539132  PMID: 16896221
22.  UAG readthrough in mammalian cells: Effect of upstream and downstream stop codon contexts reveal different signals 
Translation termination is mediated through an interaction between the release factors eRF1 and eRF3 and the stop codon within its nucleotide context. Although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved.
We have performed an in vivo analysis of translational readthrough in mouse cells in culture using a reporter system that allows the measurement of readthrough levels as low as 10-4. We first quantified readthrough frequencies obtained with constructs carrying different codons (two Gln, two His and four Gly) immediately upstream of the stop codon. There was no effect of amino acid identity or codon frequency. However, an adenine in the -1 position was always associated with the highest readthrough levels while an uracil was always associated with the lowest readthrough levels. This could be due to an effect mediated either by the nucleotide itself or by the P-site tRNA. We then examined the importance of the downstream context using eight other constructs. No direct correlation between the +6 nucleotide and readthrough efficiency was observed.
We conclude that, in mouse cells, the upstream and downstream stop codon contexts affect readthrough via different mechanisms, suggesting that complex interactions take place between the mRNA and the various components of the translation termination machinery. Comparison of our results with those previously obtained in plant cells and in yeast, strongly suggests that the mechanisms involved in stop codon recognition are conserved among eukaryotes.
PMCID: PMC29092  PMID: 11242562
23.  Eukaryotic Release Factor 3 Is Required for Multiple Turnovers of Peptide Release Catalysis by Eukaryotic Release Factor 1* 
The Journal of Biological Chemistry  2013;288(41):29530-29538.
Background: eRF3 is an essential, conserved gene, whose essential function has remained obscure.
Results: eRF3 increases multiple turnover peptide release rates beyond the level expected from its stimulation of single turnover krel.
Conclusion: eRF3 increases the efficiency of eRF1-mediated peptide release at limiting concentrations of eRF1.
Significance: This work contributes to our understanding of the essential in vivo role of eRF3.
Eukaryotic peptide release factor 3 (eRF3) is a conserved, essential gene in eukaryotes implicated in translation termination. We have systematically measured the contribution of eRF3 to the rates of peptide release with both saturating and limiting levels of eukaryotic release factor 1 (eRF1). Although eRF3 modestly stimulates the absolute rate of peptide release (∼5-fold), it strongly increases the rate of peptide release when eRF1 is limiting (>20-fold). This effect was generalizable across all stop codons and in a variety of contexts. Further investigation revealed that eRF1 remains associated with ribosomal complexes after peptide release and subunit dissociation and that eRF3 promotes the dissociation of eRF1 from these post-termination complexes. These data are consistent with models where eRF3 principally affects binding interactions between eRF1 and the ribosome, either prior to or subsequent to peptide release. A role for eRF3 as an escort for eRF1 into its fully accommodated state is easily reconciled with its close sequence similarity to the translational GTPase EFTu.
PMCID: PMC3795251  PMID: 23963452
Protein Synthesis; Ribosomes; Translation; Translation Control; Translation Release Factors; Translation; Translation Release Factors; Ribosomes; Protein Synthesis; Translational Control
24.  A Competition between Stimulators and Antagonists of Upf Complex Recruitment Governs Human Nonsense-Mediated mRNA Decay 
PLoS Biology  2008;6(4):e111.
The nonsense-mediated decay (NMD) pathway subjects mRNAs with premature termination codons (PTCs) to rapid decay. The conserved Upf1–3 complex interacts with the eukaryotic translation release factors, eRF3 and eRF1, and triggers NMD when translation termination takes place at a PTC. Contrasting models postulate central roles in PTC-recognition for the exon junction complex in mammals versus the cytoplasmic poly(A)-binding protein (PABP) in other eukaryotes. Here we present evidence for a unified model for NMD, in which PTC recognition in human cells is mediated by a competition between 3′ UTR–associated factors that stimulate or antagonize recruitment of the Upf complex to the terminating ribosome. We identify cytoplasmic PABP as a human NMD antagonizing factor, which inhibits the interaction between eRF3 and Upf1 in vitro and prevents NMD in cells when positioned in proximity to the termination codon. Surprisingly, only when an extended 3′ UTR places cytoplasmic PABP distally to the termination codon does a downstream exon junction complex enhance NMD, likely through increasing the affinity of Upf proteins for the 3′ UTR. Interestingly, while an artificial 3′ UTR of >420 nucleotides triggers NMD, a large subset of human mRNAs contain longer 3′ UTRs but evade NMD. We speculate that these have evolved to concentrate NMD-inhibiting factors, such as PABP, in spatial proximity of the termination codon.
Author Summary
The nonsense-mediated mRNA decay pathway is responsible for rapidly degrading mRNAs with premature termination codons. This is important because it prevents the production of potentially deleterious truncated proteins from aberrant mRNAs, such as those that have undergone erroneous processing. How does the cell discriminate aberrant mRNAs from those that are normal? Here we present evidence that in human cells, the targeting of an mRNA to nonsense-mediated mRNA decay depends on a competition between proteins associated with the mRNA 3′ UTR that stimulate or antagonize mRNA decay. We show that cytoplasmic poly(A)-binding protein, a protein associated with the mRNA 3′ end poly(A) tail, antagonizes mRNA decay. By contrast, a protein complex deposited onto mRNAs upon pre-mRNA splicing, called the exon junction complex, stimulates mRNA decay. Our observations suggest that the competition between these proteins, and probably other unknown proteins with similar activities, determines whether a key protein complex in the pathway, the Upf complex, is recruited to the mRNA upon translation termination, which leads to mRNA decay.
Human mRNAs with premature termination codons are detected and degraded by nonsense-mediated decay when 3' untranslated region-associated proteins, such as poly(A)-binding protein, are absent from the proximity of the terminating ribosome.
PMCID: PMC2689706  PMID: 18447585
25.  Translation Initiation Factors eIF3 and HCR1 Control Translation Termination and Stop Codon Read-Through in Yeast Cells 
PLoS Genetics  2013;9(11):e1003962.
Translation is divided into initiation, elongation, termination and ribosome recycling. Earlier work implicated several eukaryotic initiation factors (eIFs) in ribosomal recycling in vitro. Here, we uncover roles for HCR1 and eIF3 in translation termination in vivo. A substantial proportion of eIF3, HCR1 and eukaryotic release factor 3 (eRF3) but not eIF5 (a well-defined “initiation-specific” binding partner of eIF3) specifically co-sediments with 80S couples isolated from RNase-treated heavy polysomes in an eRF1-dependent manner, indicating the presence of eIF3 and HCR1 on terminating ribosomes. eIF3 and HCR1 also occur in ribosome- and RNA-free complexes with both eRFs and the recycling factor ABCE1/RLI1. Several eIF3 mutations reduce rates of stop codon read-through and genetically interact with mutant eRFs. In contrast, a slow growing deletion of hcr1 increases read-through and accumulates eRF3 in heavy polysomes in a manner suppressible by overexpressed ABCE1/RLI1. Based on these and other findings we propose that upon stop codon recognition, HCR1 promotes eRF3·GDP ejection from the post-termination complexes to allow binding of its interacting partner ABCE1/RLI1. Furthermore, the fact that high dosage of ABCE1/RLI1 fully suppresses the slow growth phenotype of hcr1Δ as well as its termination but not initiation defects implies that the termination function of HCR1 is more critical for optimal proliferation than its function in translation initiation. Based on these and other observations we suggest that the assignment of HCR1 as a bona fide eIF3 subunit should be reconsidered. Together our work characterizes novel roles of eIF3 and HCR1 in stop codon recognition, defining a communication bridge between the initiation and termination/recycling phases of translation.
Author Summary
Protein synthesis (translation) utilizes genetic information carriers, mRNAs, as templates for the production of proteins of various cellular functions. Typically it is divided into four phases: initiation, elongation, termination and ribosomal recycling. In this article we argue that the strict mechanistic separation of translation into its individual phases should be reconsidered in the light of “multitasking” of initiation factors eIF3, HCR1 and ABCE1/RLI1. In detail, we show that eIF3 and HCR1 not only promote the initiation phase but also specifically act at the other end of the translational cycle during termination. We present genetic and biochemical data linking eIF3 and HCR1 with both eukaryotic release factors (eRF1 and eRF3) and the ribosomal recycling factor ABCE1/RLI1, and propose a model for how all these factors co-operate with each other to ensure stringent selection of the stop codon. Collectively, our findings suggest that changes in one phase of translation are promptly communicated to and coordinated with changes in the other phases to maintain cellular homeostasis of all ongoing processes.
PMCID: PMC3836723  PMID: 24278036

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