Suppression of Nonsense and Frameshift Mutations Obtained by Different Methods for Inactivating the Translation Termination Factor eRF3 in Yeast Saccharomyces cerevisiae

Mutations in genes of omnipotent nonsense suppressors SUP35 and SUP45 in yeast Saccharomyces cerevisiae encoding translation termination factors eRF3 and eRF1, respectively, and prionization of the eRF3 protein may lead to the suppression of some frameshift mutations (CPC mutations). Partial inactivation of the translation termination factor eRF3 was studied in strains with unstable genetically modified prions and also in transgenic yeast S. cerevisiae strains with the substitution of the indigenous SUP35 gene for its homolog from Pichia methanolica or for a recombinant S. cerevisiae SUP35gene. It was shown that this partial inactivation leads not only to nonsense suppression, but also to suppression of the frameshift lys2-90 mutation. Possible reasons for the correlation between nonsense suppression and suppression of the CPC lys2-90 mutation and mechanisms responsible for the suppression of CPC mutations during inactivation of translation termination factors are discussed.     

Suppression of frameshift mutation as a result of partial inactivation of translation termination factors in Saccharomyces cerevisiae yeast]

Special search for frameshift mutations, which are suppressed by the cytoplasmic [PSI] factor and by omnipotent nonsense suppressors (recessive mutations in the SUP35 and SUP45 genes), partially inactivating a translation termination complex, was initiated in the LYS2 gene in the yeast Saccharomyces cerevisiae. Mutations were obtained after exposure to UV light and treatment with a mixture consisting of 1.6- and 1.8-dinitropyrene (DNP). This mixture was shown to induce mutations of the frameshift type with a high frequency. The majority of these mutations were insertions of one A or T, which is in good agreement with the data obtained in studies of DNP-induced mutagenesis in other eukaryotes. Frameshift suppression in yeast was first shown on the example of the mutation obtained in this work (lys2-90), which carried the insertion of an extra T in the sequence of five T. This frameshift suppression was shown to occur in the presence of the [PSI] factor (i.e., due to the prion form of the translation release factor eRF3) and as a result of mutations in genes SUP35 or SUP45, which partially inactivate translation termination factors eRF3 and eRF1, respectively. Alternative mechanisms of programmed translational frameshifting in the course of translation and the possibility of enhancing the effectiveness of such frameshifting in the presence of the [PSI] factor are considered.     

Partial Inactivation of Translation Termination Factors Causes Suppression of Frameshift Mutations in the Yeast Saccharomyces cerevisiae

Special search for frameshift mutations, which are suppressed by the cytoplasmic [PSI] factor and by omnipotent nonsense suppressors (recessive mutations in theSUP35and SUP45genes), partially inactivating a translation termination complex, was initiated in theLYS2gene in the yeast Saccharomyces cerevisiae.Mutations were obtained after exposure to UV light and treatment with a mixture of 1,6- and 1,8-dinitropyrene (DNP). This mixture was shown to induce mutations of the frameshift type with a high frequency. The majority of these mutations were insertions of one A or T, which is in good agreement with the data obtained in studies of DNP-induced mutagenesis in other eukaryotes. Frameshift suppression was shown on the example of the mutation obtained in this work (lys2-90), which carried the insertion of an extra T in the sequence of five T. This frameshift suppression was first shown to occur in the presence of the [PSI] factor (i.e., due to the prionization of the translation release factor eRF3) and as a result of mutations in genes SUP35orSUP45, which partially inactivate translation termination factors eRF3 and eRF1, respectively. Alternative mechanisms of programmed translational frameshifting in the course of translation and the possibility of enhancing the effectiveness of such frameshifting in the presence of the [PSI] factor are considered.     

Increased tRNA concentration in yeast containing mutant termination translation factors eRF1 and eRF3

Earlier we have characterized strains bearing mutations in essential genes SUP45 and SUP35 of yeast S. cerevisiae, encoding translation termination factors eRF1 and eRF3 respectively. In the present work nonsense-mutants on genes SUP45 and SUP35 have been compared by a level of eight tRNA: tRNATyr, tRNAGln, tRNATrp, tRNALeu and tRNAArg (previously described as potentially suppressor tRNA), and also tRNAPro, tRNAHis and tRNAGly. We have not revealed preferable increase in amount of natural suppressor tRNA. The majority of the investigated mutations leads to increase in a level of all investigated tRNA. The mechanisms providing viability of nonsense-mutants on essential genes SUP45 and SUP35 are discussed.     

Conservation of the MC domains in eukaryotic termination factor eRF3

Eukaryotic translation termination employs two protein factors, eRF1 and eRF3. Proteins of the eRF3 family each consist of three domains. The N and M domains vary in different species, while the C domains are highly homologous. The MC domains of Homo sapiens eRF3a (hGSPT I), Xenopus laevis eRF3 (XSup35), and Mus musculus eRF3a (mGSPTI) and eRF3b (mGSPT2) were found to compensate for the sup35-21(ts) temperature-sensitive mutation and lethal disruption of the SUP35 gene in yeast Saccharomyces cerevisiae. At the same time, strains containing the MC domains of the eRF3 proteins from different species differed in growth rate and the efficiency of translation termination.     

Nonsense mutations in the essential gene<Emphasis Type="Italic"> SUP35</Emphasis> of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> are non-lethal

In the present work we have characterized for the first time non-lethal nonsense mutations in the essential gene SUP35, which codes for the translation termination factor eRF3 in Saccharomyces cerevisiae. The screen used was based on selection for simultaneous suppression of two auxotrophic nonsense mutations. Among 48 mutants obtained, sixteen were distinguished by the production of a reduced amount of eRF3, suggesting the appearance of nonsense mutations. Fifteen of the total mutants were sequenced, and the presence of nonsense mutations was confirmed for nine of them. Thus a substantial fraction of the sup35 mutations recovered are nonsense mutations located in different regions of SUP35, and such mutants are easily identified by the fact that they express reduced amounts of eRF3. Nonsense mutations in the SUP35 gene do not lead to a decrease in levels of SUP35 mRNA and do not influence the steady-state level of eRF1. The ability of these mutations to complement SUP35 gene disruption mutations in different genetic backgrounds and in the absence of any tRNA suppressor mutation was demonstrated. The missense mutations studied, unlike nonsense mutations, do not decrease steady-state amounts of eRF3.Communicated by C. P. Hollenberg     

Viable nonsense mutants for the SUP45 gene in the yeast Saccharomyces cerevisiae are lethal at increased temperature

Nonlethal nonsense mutations obtained earlier in the essential gene SUP45 encoding the translation termination eRFI factor in the yeast Saccharomyces cerevisiae were further characterized. Strains carrying these mutations retain the viability, since the full-length eRF1 protein is present in these strains, although in decreased amounts as compared to wild-type cells, together with a truncated eRF1. All nonsense mutations are likely to be located in a weak termination context, because a change in the stop codon UGAA (in the case of mutation sup45-107) to UAGA (sup45-107.2) led to the alteration of the local context from a weak to strong and to the lethality of the strain carrying sup45-107.2. All nonsense mutations studied are characterized by thermosensitivity expressed as cell mortality after cultivation at 37 degrees C. When grown under nonpermissive conditions (37 degrees C), cells of nonsense mutants sup45-104, sup45-105. and sup45-107 display a decrease in the amount of the truncated eRF1 protein without reduction in the amount of the full-length eRF1 protein. The results of this study suggest that the N-terminal eRF1 fragment is indispensable for cell viability of nonsense mutants due to the involvement in termination of translation.     

Molecular population genetics and evolution of a prion-like protein in Saccharomyces cerevisiae

The prion-like behavior of Sup35p, the eRF3 homolog in the yeast Saccharomyces cerevisiae, mediates the activity of the cytoplasmic nonsense suppressor known as [PSI(+)]. Sup35p is divided into three regions of distinct function. The N-terminal and middle (M) regions are required for the induction and propagation of [PSI(+)] but are not necessary for translation termination or cell viability. The C-terminal region encompasses the termination function. The existence of the N-terminal region in SUP35 homologs of other fungi has led some to suggest that this region has an adaptive function separate from translation termination. To examine this hypothesis, we sequenced portions of SUP35 in 21 strains of S. cerevisiae, including 13 clinical isolates. We analyzed nucleotide polymorphism within this species and compared it to sequence divergence from a sister species, S. paradoxus. The N domain of Sup35p is highly conserved in amino acid sequence and is highly biased in codon usage toward preferred codons. Amino acid changes are under weak purifying selection based on a quantitative analysis of polymorphism and divergence. We also conclude that the clinical strains of S. cerevisiae are not recently derived and that outcrossing between strains in S. cerevisiae may be relatively rare in nature.     

The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae.

The product of the yeast SUP45 gene (Sup45p) is highly homologous to the Xenopus eukaryote release factor 1 (eRF1), which has release factor activity in vitro. We show, using the two-hybrid system, that in Saccharomyces cerevisiae Sup45p and the product of the SUP35 gene (Sup35p) interact in vivo. The ability of Sup45p C-terminally tagged with (His)6 to specifically precipitate Sup35p from a cell lysate was used to confirm this interaction in vitro. Although overexpression of either the SUP45 or SUP35 genes alone did not reduce the efficiency of codon-specific tRNA nonsense suppression, the simultaneous overexpression of both the SUP35 and SUP45 genes in nonsense suppressor tRNA-containing strains produced an antisuppressor phenotype. These data are consistent with Sup35p and Sup45p forming a complex with release factor properties. Furthermore, overexpression of either Xenopus or human eRF1 (SUP45) genes also resulted in anti-suppression only if that strain was also overexpressing the yeast SUP35 gene. Antisuppression is a characteristic phenotype associated with overexpression of both prokaryote and mitochondrial release factors. We propose that Sup45p and Sup35p interact to form a release factor complex in yeast and that Sup35p, which has GTP binding sequence motifs in its C-terminal domain, provides the GTP hydrolytic activity which is a demonstrated requirement of the eukaryote translation termination reaction.     

Frameshift suppression through inactivation of translation termination in yeast Saccharomyces cerevisiae: significance of the local context

Site-directed mutagenesis and nucleotide sequence analysis were used to study the roles of the global and local contexts in suppression of the lys2-90 frameshift (FS) mutation in Saccharomyces cerevisiae. Global context features established for the LYS2 mRNA region containing the extra T (lys2-90) were similar to those characteristic of regions involved in translational frameshifting. These were a potential ability of the region to form a pseudoknot and the presence of heptanucleotide CUU UGA C with the "hungry" UGA nonsense codon in the pseudoknot. Some local context features proved to be essential for the phenotypic expression of FS suppression as a result of translational frameshifting. Two amino acid substitutions determined by the nucleotide sequence between the extra U and the UGA nonsense codon lacked expression. A dependence was observed between the efficiency of FS suppression and the type of the nonsense codon located at a particular position downstream of the extra nucleotide (UGA > UAG > UAA). When translation termination was inactivated, nonsense suppression and FS suppression correlated with each other. These results suggest that translational frameshifting, which underlies suppression in the case of inactivation of translation termination, most likely takes place on the nonsense codon arising as a result of insertion of an extra nucleotide.     

Phenotypic manifestation of epigenetic determinant [ISP+] in Saccharomyces serevisiae depends on combination of mutations in SUP35 and SUP45 genes

It is known that translation fidelity in Saccharomyces yeast is determined by factors of genetic and epigenetic (prion) nature. The work represents results of further analysis of strains containing non-chromosomal determinant [ISP+], described earlier. This determinant is involved in the control of translation fidelity and some of its properties indicate that it is a prion. [ISP+] manifests phenotypically as antisuppressor of two sup35 mutations and can be cured by guanidine hydrochloride (GuHCl). Here we have shown that sup35 mutants containing [ISP+] contain also additional sup45 mutations. These mutations cause amino acid replacements in different regions of eRF1 translation termination factor, encoded by SUP45 gene. Strains bearing sup35-25 mutation contain sup45 mutation, which causes amino acid replacement at position 400 of eRF1; strains bearing sup35-10 contain mutation causing replacement, which alters eRF1 at position 75. Thus, antisuppressor phenotype of [ISP+] strains depends on interaction of sup35 and sup45 mutations, as well as on the GuHCl-curable epigenetic determinant.     

Yeast polypeptide chain release factors eRF1 and eRF3 are involved in cytoskeleton organization and cell cycle regulation

Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. eRF1 recognizes nonsense codons UAA, UAG, and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRF1-dependent manner. In the yeast Saccharomyces cerevisiae, eRF1 and eRF3 are encoded by the SUP45 and SUP35 genes, respectively. Here we show that in yeast shortage of any one of the release factors was accompanied by a reduction in the levels of the other release factor and resulted in a substantial increase of nonsense codon readthrough. Besides, repression of the genes encoding these factors caused different effects on cell morphology. Repression of the SUP35 gene caused accumulation of cells of increased size with large buds. This was accompanied by the disappearance of actin cytoskeletal structures, impairment of the mitotic spindle structure, and defects in nuclei division and segregation in mitosis. The evolutionary conserved C-terminal domain of eRF3 similar to the elongation factor EF-1alpha was responsible for these effects. Repression of the SUP45 gene caused accumulation of unbudded cells with 2C and higher DNA content, indicating that DNA replication is uncoupled from budding. The data obtained suggest that eRF1 and eRF3 play additional, nontranslational roles in the yeast cell.     

Eukaryotic release factors (eRFs) history

In the present review, we describe the history of the identification of the eukaryotic translation termination factors eRF1 and eRF3. As in the case of several proteins involved in general and essential processes in all cells (e.g., DNA replication, gene expression regulation.) the strategies and methodologies used to identify these release factors were first established in prokaryotes. The genetic investigations in Saccharomyces cerevisiae have made a major contribution in the field. A large amount of data have been produced, from which it was concluded that the SUP45 and SUP35 genes were controlling translation termination but were also involved in other functions important for the cell organization and the cell cycle accomplishment. This does not seem to be restricted to yeast but is also probably the case in eukaryotes in general. The biochemical studies of the proteins encoded by the higher eukaryote homologs of SUP45 and SUP35 were efficient and permitted the identification of eRF1 as being the key protein in the termination process, eRF3 having a stimulating role. Around 25 years were needed after the identification of sup45 and sup35 mutants for the characterization of their gene products as eRF1 and eRF3, respectively. It also has to be pointed out that if the results came first from bacteria, the identification of RF3 and eRF3 was made practically at the same time. Moreover, eRF1 was the first crystal structure obtained for a class-1 release factor, the bacterial RF2 structure came later. The goal is now to understand at the molecular level the roles of both eRF1 and eRF3 in addition to their translation termination functions.     

Polymorphism of the SUP35 gene and its product in the Saccharomyces cerevisiae yeasts

The product of the SUP35 gene of the saccharomycete yeast, the translation termination eRF3 factor, can be converted in prion, the heritable determinant of protein nature. The nucleotide sequence of this gene from the strain belonging to Peterhof genetic lines of the yeast Saccharomyces cerevisiae was determined. A comparison of the identified sequence with SUP35 sequences in the database of GenBank allowed the detection of polymorphic sites both in the SUP35 gene and its product. The location of polymorphic sites in the evolutionarily nonconserved N-terminal protein region confirmed that this eRF3 fragment lacks functions vital to life activity. Nevertheless, these sites are located in the vicinity of sites, whose role in the prion conversion of eRF3 has been established. Based on this, natural polymorphism of the primary eRF3 structure is assumed to be connected with the existence of different variants (strains) of its prion analog.     

Overexpression of genes encoding tRNA<Superscript>Tyr</Superscript> and tRNA<Superscript>Gln</Superscript> increases the viability of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains with nonsense mutations in the <Emphasis Type="Italic">SUP45</Emphasis> gene

At present, the machinery supporting the viability of organisms possessing nonsense mutations in essential genes is not entirely understood. Nonsense mutants of Saccharomyces cerevisiae yeast containing a premature translation termination codon in the essential SUP45 gene are known. These strains are viable in the absence of mutant suppressor tRNAs; hence, the existence of alternative mechanisms providing nonsense suppression and mutant viability is conjectured. Analysis of clones obtained by transformation of a strain bearing a nonsense-mutant allele of SUP45 with a multicopy yeast genomic library revealed three genes encoding wild-type tRNA^Tyr and four genes encoding wild-type tRNA^Gln, which increased nonsense mutant viability. Moreover, overexpression of these genes leads to an increase in the amount of the full-length eRF1 protein in cells and compensates for heat sensitivity in the nonsense mutants. Probable ways of tRNA^Tyr and tRNA^Gln influence on the increase in the viability of strains with nonsense mutations in SUP45 are discussed.     

Viable nonsense mutants for the <Emphasis Type="Italic">SUP45</Emphasis> gene in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> are lethal at increased temperature

Nonlethal nonsense mutations obtained earlier in the essential gene SUP45 encoding the translation termination factor eRF1 in the yeast Saccharomyces cerevisiae were further characterized. Strains carrying these mutations retain the viability, since the full-length eRF1 protein is present in these strains, although in decreased amounts as compared to wild-type cells, together with a trucated eRF1. All nonsense mutations are likely to be located in a weak termination context, because a change in the stop codon UGAA (in the case of mutation sup45-107) to UAGA (sup45-107.2) led to the alteration of the local context from a weak to strong and to the lethality of the strain carrying sup45-107.2. All nonsense mutations studied are characterized by thermosensitivity expressed as cell mortality after cultivation at 37°C. When grown under nonpermissive conditions (37°C), cells of nonsense mutants sup45-104, sup45-105, and sup45-107 display a decrease in the amount of the truncated eRF1 protein without reduction in the amount of the full-length eRF1 protein. The results of this study suggest that the N-terminal eRF1 fragment is indispensable for cell viability of nonsense mutants due to the involvement in termination of translation.     

Conservation of the MC domains in eukaryotic release factor eRF3

Eukaryotic translation termination employs two protein factors, eRF1 and eRF3. Proteins of the eRF3 family each consist of three domains. The N and M domains vary in different species, while the C domains are highly homologous. The MC domains of Homo sapiens eRF3a (hGSPT1), Xenopus laevis eRF3 (XSup35), and Mus musculus eRF3a (mGSPT1) and eRF3b (mGSPT2) were found to compensate for the sup35-21(ts) temperature-sensitive mutation and lethal disruption of the SUP35 gene in yeast Saccharomyces cerevisiae. At the same time, strains containing the MC domains of the eRF3 proteins from different species differed in growth rate and the efficiency of translation termination.     

The role of Upf proteins in modulating the translation read-through of nonsense-containing transcripts

The yeast UPF1, UPF2 and UPF3 genes encode trans-acting factors of the nonsense-mediated mRNA decay pathway. In addition, the upf1Delta strain demonstrates a nonsense suppression phenotype and Upf1p has been shown to interact with the release factors eRF1 and eRF3. In this report, we show that both upf2Delta and upf3Delta strains demonstrate a nonsense suppression phenotype independent of their effect on mRNA turnover. We also demonstrate that Upf2p and Upf3p interact with eRF3, and that their ability to bind eRF3 correlates with their ability to complement the nonsense suppression phenotype. In vitro experiments demonstrate that Upf2p, Upf3p and eRF1 compete with each other for interacting with eRF3. Con versely, Upf1p binds to a different region of eRF3 and can form a complex with these factors. These results suggest a sequential surveillance complex assembly pathway, which occurs during the premature translation termination process. We propose that the observed nonsense suppression phenotype in the upfDelta strains can be attributed to a defect in the surveillance complex assembly.