Modulation of efficiency of translation termination in Saccharomyces cerevisiae

Nonsense suppression is a readthrough of premature termination codons. It typically occurs either due to the recognition of stop codons by tRNAs with mutant anticodons, or due to a decrease in the fidelity of translation termination. In the latter case, suppressors usually promote the readthrough of different types of nonsense codons and are thus called omnipotent nonsense suppressors. Omnipotent nonsense suppressors were identified in yeast Saccharomyces cerevisiae in 1960s, and most of subsequent studies were performed in this model organism. Initially, omnipotent suppressors were localized by genetic analysis to different protein- and RNA-encoding genes, mostly the components of translational machinery. Later, nonsense suppression was found to be caused not only by genomic mutations, but also by epigenetic elements, prions. Prions are self-perpetuating protein conformations usually manifested by infectious protein aggregates. Modulation of translational accuracy by prions reflects changes in the activity of their structural proteins involved in different aspects of protein synthesis. Overall, nonsense suppression can be seen as a “phenotypic mirror” of events affecting the accuracy of the translational machine. However, the range of proteins participating in the modulation of translation termination fidelity is not fully elucidated. Recently, the list has been expanded significantly by findings that revealed a number of weak genetic and epigenetic nonsense suppressors, the effect of which can be detected only in specific genetic backgrounds. This review summarizes the data on the nonsense suppressors decreasing the fidelity of translation termination in S. cerevisiae, and discusses the functional significance of the modulation of translational accuracy.     

Suppression of premature termination codons as a therapeutic approach

In this review, we describe our current understanding of translation termination and pharmacological agents that influence the accuracy of this process. A number of drugs have been identified that induce suppression of translation termination at in-frame premature termination codons (PTCs; also known as nonsense mutations) in mammalian cells. We discuss efforts to utilize these drugs to suppress disease-causing PTCs that result in the loss of protein expression and function. In-frame PTCs represent a genotypic subset of mutations that make up ~11% of all known mutations that cause genetic diseases, and millions of patients have diseases attributable to PTCs. Current approaches aimed at reducing the efficiency of translation termination at PTCs (referred to as PTC suppression therapy) have the goal of alleviating the phenotypic consequences of a wide range of genetic diseases. Suppression therapy is currently in clinical trials for treatment of several genetic diseases caused by PTCs, and preliminary results suggest that some patients have shown clinical improvements. While current progress is promising, we discuss various approaches that may further enhance the efficiency of this novel therapeutic approach.     

Suppression of premature termination codons as a therapeutic approach

In this review, we describe our current understanding of translation termination and pharmacological agents that influence the accuracy of this process. A number of drugs have been identified that induce suppression of translation termination at in-frame premature termination codons (PTCs; also known as nonsense mutations) in mammalian cells. We discuss efforts to utilize these drugs to suppress disease-causing PTCs that result in the loss of protein expression and function. In-frame PTCs represent a genotypic subset of mutations that make up ~11% of all known mutations that cause genetic diseases, and millions of patients have diseases attributable to PTCs. Current approaches aimed at reducing the efficiency of translation termination at PTCs (referred to as PTC suppression therapy) have the goal of alleviating the phenotypic consequences of a wide range of genetic diseases. Suppression therapy is currently in clinical trials for treatment of several genetic diseases caused by PTCs, and preliminary results suggest that some patients have shown clinical improvements. While current progress is promising, we discuss various approaches that may further enhance the efficiency of this novel therapeutic approach.     

Readthrough compounds for nonsense mutations: bridging the translational gap

Approximately 10% of all pathological mutations are nonsense mutations that are responsible for several severe genetic diseases for which no treatment regimens are currently available. The most widespread strategy for treating nonsense mutations is by enhancing ribosomal readthrough of premature termination codons (PTCs) to restore the production of the full-length protein. In the past decade several compounds with readthrough potential have been identified. However, although preclinical results on these compounds are promising, clinical studies have not yielded positive outcomes. We review preclinical and clinical research related to readthrough compounds and characterize factors that contribute to the observed translational gap.     

Aminoglycosides and other factors promoting stop codon readthrough in human cells

Enhanced stop codon readthrough is a potential treatment strategy for diseases caused by nonsense mutations. Here, we compare readthrough levels induced by three types of factors: aminoglycoside antibiotics, suppressor tRNAs, and factors decreasing translation termination efficiency. We show that the highest levels of readthrough were obtained by prolonged treatment with aminoglycosides and suppressor tRNAs, whereas prolonged depletion of release factors induced only a moderate increase in readthrough. We discuss the benefits and inconvenients of the three types of factors for their use in the therapy of diseases caused by premature stop codons.     

Construction and testing of a bacterial luciferase reporter gene system forin Vivo measurement of nonsense suppression inStreptomyces

A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.     

Mutations in eukaryotic release factors 1 and 3 act as general nonsense suppressors in Drosophila

In a screen for suppressors of the Drosophila wingless(PE4) nonsense allele, we isolated mutations in the two components that form eukaryotic release factor. eRF1 and eRF3 comprise the translation termination complex that recognizes stop codons and catalyzes the release of nascent polypeptide chains from ribosomes. Mutations disrupting the Drosophila eRF1 and eRF3 show a strong maternal-effect nonsense suppression due to readthrough of stop codons and are zygotically lethal during larval stages. We tested nonsense mutations in wg and in other embryonically acting genes and found that different stop codons can be suppressed but only a subset of nonsense alleles are subject to suppression. We suspect that the context of the stop codon is significant: nonsense alleles sensitive to suppression by eRF1 and eRF3 encode stop codons that are immediately followed by a cytidine. Such suppressible alleles appear to be intrinsically weak, with a low level of readthrough that is enhanced when translation termination is disrupted. Thus the eRF1 and eRF3 mutations provide a tool for identifying nonsense alleles that are leaky. Our findings have important implications for assigning null mutant phenotypes and for selecting appropriate alleles to use in suppressor screens.     

Discrimination between defects in elongation fidelity and termination efficiency provides mechanistic insights into translational readthrough

The suppression of stop codons (termed translational readthrough) can be caused by a decreased accuracy of translation elongation or a reduced efficiency of translation termination. In previous studies, the inability to determine the extent to which each of these distinct processes contributes to a readthrough phenotype has limited our ability to evaluate how defects in the translational machinery influence the overall termination process. Here, we describe the combined use of misincorporation and readthrough reporter systems to determine which of these mechanisms contributes to translational readthrough in Saccharomyces cerevisiae. The misincorporation reporter system was generated by introducing a series of near-cognate mutations into functionally important residues in the firefly luciferase gene. These constructs allowed us to monitor the incidence of elongation errors by monitoring the level of firefly luciferase activity from a mutant allele inactivated by a single missense mutation. In this system, an increase in luciferase activity should reflect an increased level of misincorporation of the wild-type amino acid that provides an estimate of the overall fidelity of translation elongation. Surprisingly, we found that growth in the presence of paromomycin stimulated luciferase activity for only a small subset of the mutant proteins examined. This suggests that the ability of this aminoglycoside to induce elongation errors is limited to a subset of near-cognate mismatches. We also found that a similar bias in near-cognate misreading could be induced by the expression of a mutant form of ribosomal protein (r-protein) S9B or by depletion of r-protein L12. We used this misincorporation reporter in conjunction with a readthrough reporter system to show that alterations at different regions of the ribosome influence elongation fidelity and termination efficiency to different extents.     

Evidence that the supE44 Mutation of Escherichia coli Is an Amber Suppressor Allele of glnX and that It Also Suppresses Ochre and Opal Nonsense Mutations

Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.Translation termination is mediated by one of the three stop codons (UAA, UAG, or UGA). When such stop codons arise in coding sequences due to mutations, referred to as nonsense mutations, they lead to abrupt arrest of the translation process. However, the termination efficiency of such nonsense codons is not 100%, as certain tRNAs have the ability to read these nonsense codons. Genetic code ambiguity is seen in several organisms. Stop codons have been shown to have alternate roles apart from translation termination. In organisms from all three domains of life, UGA encodes selenocysteine through a specialized mechanism. In Methanosarcinaceae, UAG encodes pyrrolysine (3). UAA and UAG are read as glutamine codons in some green algae and ciliates such as Tetrahymena and Diplomonads (24), and UAG alone encodes glutamine in Moloney murine leukemia virus (32). UGA encodes cysteine in Euplotes; tryptophan in some ciliates, Mycoplasma species, Spiroplasma citri, Bacillus, and tobacco rattle virus; and an unidentified amino acid in Pseudomicrothorax dubius and Nyctotherus ovalis (30). In certain cases the context of the stop codon in translational readthrough has been shown to play a role; for example, it has been reported that in vitro in tobacco mosaic virus, UAG and UAA are misread by tRNA^Tyr in a highly context-dependent manner (34, 9).Termination suppressors are of three types, i.e., amber, ochre, and opal suppressors, which are named based on their ability to suppress the three stop codons. Amber suppressors can suppress only amber codons, whereas ochre suppressors can suppress ochre codons (by normal base pairing) as well as amber codons (by wobbling) and opal suppressors can read opal and UGG tryptophan codon in certain cases. As described by Sambrook et al. (27), a few amber suppressors can also suppress ochre mutations by wobbling. The suppression efficiency varies among these suppressors, with amber suppressors generally showing increased efficiency over ochre and opal suppressors. supE44, an amber suppressor tRNA, is an allele of and is found in many commonly used strains of Escherichia coli K-12. Earlier studies have shown that supE44 is a weak amber suppressor and that its efficiency varies up to 35-fold depending on the reading context of the stop codon (8).Translational accuracy depends on several factors, which include charging of tRNAs with specific amino acids, mRNA decoding, and the presence of antibiotics such as streptomycin and mutations in ribosomal proteins which modulate the fidelity of the translational machinery. Among these, mRNA decoding errors have been reported to occur at a frequency ranging from about 10⁻³ to 10⁻⁴ per codon. Translational misreading errors also largely depend on the competition between cognate and near-cognate tRNA species. Poor availability of cognate tRNAs increases misreading (18).Several studies with E. coli and Saccharomyces cerevisiae have shown the readthrough of nonsense codons in suppressor-free cells. In a suppressor-free E. coli strain, it has been shown in vitro that glutamine is incorporated at the nonsense codons UAG and UAA (26). It has been reported that overexpression of wild-type tRNA^Gln in yeast suppresses amber as well as ochre mutations (25). In this study, we have confirmed the presence of an amber suppressor mutation in the glnX gene in a supE44 strain by sequence analysis. This was done essentially because we observed that supE44 could also suppress lacZ ochre mutations, albeit inefficiently. On further investigation using an in vivo luciferase reporter assay system for tRNA-mediated nonsense suppression (28), we found that the efficiency of suppression of amber lesion by supE44 is significantly higher than that reported previously in the literature. An increased ability to suppress ochre and opal nonsense mutations was observed in cells bearing supE44 compared to in the wild type. Such an effect was observed only in the stationary phase and was abolished in logarithmic phase.     

The paradox of viable <Emphasis Type="Italic">sup45</Emphasis> STOP mutations: a necessary equilibrium between translational readthrough,activity and stability of the protein

The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q → TAA, 242R → TGA, 317L → TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells.     

The influence of the reading context upon the suppression of nonsense codons

Summary We have examined the response of phage T4 nonsense mutations located at various sites within the same cistron to different suppression agents. A wide range of suppression efficiency is found for both ochre (UAA) and amber (UAG) mutations under conditions where suppression provides a measurement of the amount of chain propagation past the mutated site. We have established a relationship between our measurement-the size of the phage yield-and the amount of rIIB product present in the infection. Our data suggest that the 1000-fold range of variations in yields observed in the rIIB cistron corresponds to a 30-fold range of variation in the level of rIIB product, i.e. in the relative frequency of chain propagation past the various nonsense codons included in our test.From the parallelism of response of any particular mutant to very different suppression mechanisms we conclude that the efficiency of suppression is site specific, that is to say, that the main factor determining the frequency of chain propagation at a nonsense codon by any type of suppression mechanism is the nucleotide sequence adjacent to the nonsense codon (reading context).We propose that the recognition of a natural termination signal involves a sequence longer than a nonsense codon and that nonsense codons outside of their natural environment induce variable termination rates which are reflected in the suppression potential.     

An rRNA Fragment and Its Antisense Can Alter Decoding of Genetic Information

rRNA plays a central role in protein synthesis and is intimately involved in the initiation, elongation, and termination stages of translation. However, the mode of its participation in these reactions, particularly as to the decoding of genetic information, remains elusive. In this paper, we describe a new approach that allowed us to identify an rRNA segment whose function is likely to be related to translation termination. By screening an expression library of random rRNA fragments, we identified a fragment of the Escherichia coli 23S rRNA (nucleotides 74 to 136) whose expression caused readthrough of UGA nonsense mutations in certain codon contexts in vivo. The antisense RNA fragment produced a similar effect, but in neither case was readthrough of UAA or UAG observed. Since termination at UGA in E. coli specifically requires release factor 2 (RF2), our data suggest that the fragments interfere with RF2-dependent termination.     

Ribosomal RNAs in translation termination: facts and hypotheses

It is now well established that ribosomal RNAs (rRNAs) play an active role in every aspect of translation. This review focuses on recent evidence for the involvement of rRNAs from both subunits of the ribosome in translation termination. This evidence comprises data obtained with rRNA mutants both in vivo and in vitro. In particular, mutations in specific regions of rRNAs caused readthrough of nonsense codons in vivo. Consistent with their in vivo characteristics, the mutations decreased the productive association of the ribosome with release factor 2 (RF2) and the efficiency of catalysis of peptidyl-tRNA hydrolysis in the presence of RF2 in realistic in vitro termination systems. It is now evident that genetic selections for termination-defective mutants in vivo and their characterization in realistic in vitro termination assays will rapidly advance our understanding of the mechanism of termination.     

Allele-specific therapy: suppression of nonsense mutations by readthrough inducers

Ten percent of human hereditary diseases are linked to nonsense mutations (premature termination codon). These mutations lead to premature translation termination, trigger the synthesis of a truncated protein and possibly lead to mRNA degradation by the NMD pathway (nonsense mediated mRNA decay). For the past ten years, therapeutic strategies have emerged which attempt to use molecules that facilitate tRNA incorporation at premature stop codon (readthrough), thus allowing for the synthesis of a full length protein. Molecules currently used for this approach are mostly aminoglycoside antibiotics (gentamicin, amikacin…) that bind the decoding center of the ribosome. This therapeutic approach has been studied for various genetic diseases including Duchenne muscular dystrophy (DMD) and cystic fibrosis. The feasibility of this approach depends on induced readthrough level, mRNA quantity, re-expressed protein functionality and characteristics of each disease.     

Mutational eidence for a functional connection between two domains of 23S rRNA in translation termination

Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.     

A simple and sensitive in vivo luciferase assay for tRNA-mediated nonsense suppression.

We present a rapid assay for tRNA suppression in living Escherichia coli. An amber, ochre, or opal nonsense mutation in a cloned luxB gene from the bacterium Vibrio harveyi was suppressed. Because luciferase (Lux) activity depends completely on the appearance of the full-length luxB gene product, the amount of light produced was proportional to tRNA-mediated nonsense suppression in the cell. This luminometric assay was notably quicker, easier, and more sensitive than a traditional colorimetric assay employing beta-galactosidase. Assays required only one addition to a growing culture and were complete within 1 min. Light output was directly proportional to the amount of bacterial luciferase in a sample over a range of greater than or equal to 40,000-fold. Fewer than 100 cells were required for detection of Lux with ordinary instrumentation; assays were 80-fold more sensitive than simultaneous beta-galactosidase measurements. Assayed cells survived and could be recovered as colony formers. The beta-galactosidase colorimetric assay and the luciferase assay were similarly reproducible. Light from colonies expressing Lux was visible to the dark-adapted eye and useful for screening. A rapid assay that does not depend on the formation of permanent transformants can be based on electroporation followed by luminometry.     

Translational fidelity mutations in 18S rRNA affect the catalytic activity of ribosomes and the oxidative balance of yeast cells

The function of mutations rdn1A, rdn1T, and rdn2 in 18S rRNA of Saccharomyces cerevisiae is investigated. The mutations correspond to substitutions C1054A, C1054U in helix 34, and G517A in helix 18 of 16S rRNA in Escherichia coli, respectively, in which the first and third mutations caused nonsense suppression, while C1054U caused no suppression. In yeast, rdn1A caused phenotypic suppression at nonsense codons, whereas rdn1T and rdn2 caused antisuppression. We provide in vitro evidence that, in addition, rdn1A decreases translational accuracy at sense codons as well, by a factor of 8, accompanied by extreme sensitivity to paromomycin, compatible with its error-prone character. Mutations rdn1T andrdn2 exhibit hyperaccuracy and paromomycin resistance. Thus, mutations in conserved rRNA regions may affect the same functions in the various species but in opposite directions. Mutation rdn1A, but not rdn1T or rdn2, affected also the catalytic activity of the ribosome, a 60S subunit activity. The rate of peptide bond formation was reduced to half its normal value, indicating a communication between the two subunits. Moreover, error-prone mutation rdn1A was less susceptible to oxidative modifications than wild type, indicated by decreased lipid peroxidation and nonprotein/protein disulfides, as well as by increased protein thiols. In contrast, hyperaccurate mutations rdn1T and rdn2 displayed increased oxidative stress. Our results suggest that the cells may consume more energy to achieve hyperaccuracy leading to increased oxidative modifications.     

Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae

Background

Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl – dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough.

Results

We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p.

Conclusions

The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.     

Mutations to nonsense codons in human genetic disease: implications for gene therapy by nonsense suppressor tRNAs.

Nonsense suppressor tRNAs have been suggested as potential agents for human somatic gene therapy. Recent work from this laboratory has described significant effects of 3' codon context on the efficiency of human nonsense suppressors. A rapid increase in the number of reports of human diseases caused by nonsense codons, prompted us to determine how the spectrum of mutation to either UAG, UAA or UGA codons and their respective 3' contexts, might effect the efficiency of human suppressor tRNAs employed for purposes of gene therapy. This paper presents a survey of 179 events of mutations to nonsense codons which cause human germline or somatic disease. The analysis revealed a ratio of approximately 1:2:3 for mutation to UAA, UAG and UGA respectively. This pattern is similar, but not identical, to that of naturally occurring stop codons. The 3' contexts of new mutations to stop were also analysed. Once again, the pattern was similar to the contexts surrounding natural termination signals. These results imply there will be little difference in the sensitivity of nonsense mutations and natural stop codons to suppression by nonsense suppressor tRNAs. Analysis of the codons altered by nonsense mutations suggests that efforts to design human UAG suppressor tRNAs charged with Trp, Gln, and Glu; UAA suppressors charged with Gln and Glu, and UGA suppressors which insert Arg, would be an essential step in the development of suppressor tRNAs as agents of human somatic gene therapy.