Efficiency Of Nonsense Suppression Research Articles

Haploid yeast cells undergo a reversible phenotypic switch associated with chromosome II copy number.

Background SUP35 and SUP45 are essential genes encoding polypeptide chain release factors. However, mutants for these genes may be viable but display pleiotropic phenotypes which include, but are not limited to, nonsense suppressor phenotype due to translation termination defect. [PSI +] prion formation is another Sup35p-associated mechanism leading to nonsense suppression through decreased availability of functional Sup35p. [PSI +] differs from genuine sup35 mutations by the possibility of its elimination and subsequent re-induction. Some suppressor sup35 mutants had also been shown to undergo a reversible phenotypic switch in the opposite direction. This reversible switching had been attributed to a prion termed [ISP +]. However, even though many phenotypic and molecular level features of [ISP +] were revealed, the mechanism behind this phenomenon has not been clearly explained and might be more complex than suggested initially.ResultsHere we took a genomic approach to look into the molecular basis of the difference between the suppressor (Isp−) and non-suppressor (Isp+) phenotypes. We report that the reason for the difference between the Isp+ and the Isp− phenotypes is chromosome II copy number changes and support our finding with showing that these changes are indeed reversible by reproducing the phenotypic switch and tracking karyotypic changes. Finally, we suggest mechanisms that mediate elevation in nonsense suppression efficiency upon amplification of chromosome II and facilitate switching between these states.Conclusions(i) In our experimental system, amplification of chromosome II confers nonsense suppressor phenotype and guanidine hydrochloride resistance at the cost of overall decreased viability in rich medium. (ii) SFP1 might represent a novel regulator of chromosome stability, as SFP1 overexpression elevates frequency of the additional chromosome loss in our system. (iii) Prolonged treatment with guanidine hydrochloride leads to selection of resistant isolates, some of which are disomic for chromosome II.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0464-4) contains supplementary material, which is available to authorized users.

Chromosome VIII disomy influences the nonsense suppression efficiency and transition metal tolerance of the yeast Saccharomyces cerevisiae.

The SUP35 gene of the yeast Saccharomyces cerevisiae encodes the translation termination factor eRF3. Mutations in this gene lead to the suppression of nonsense mutations and a number of other pleiotropic phenotypes, one of which is impaired chromosome segregation during cell division. Similar effects result from replacing the S. cerevisiae SUP35 gene with its orthologues. A number of genetic and epigenetic changes that occur in the sup35 background result in partial compensation for this suppressor effect. In this study we showed that in S. cerevisiae strains in which the SUP35 orthologue from the yeast Pichia methanolica replaces the S. cerevisiae SUP35 gene, chromosome VIII disomy results in decreased efficiency of nonsense suppression. This antisuppressor effect is not associated with decreased stop codon read-through. We identified SBP1, a gene that localizes to chromosome VIII, as a dosage-dependent antisuppressor that strongly contributes to the overall antisuppressor effect of chromosome VIII disomy. Disomy of chromosome VIII also leads to a change in the yeast strains' tolerance of a number of transition metal salts.

Enhanced Yield of Recombinant Proteins with Site-Specifically Incorporated Unnatural Amino Acids Using a Cell-Free Expression System

Using a commercial protein expression system, we sought the crucial elements and conditions for the expression of proteins with genetically encoded unnatural amino acids. By identifying the most important translational components, we were able to increase suppression efficiency to 55% and to increase mutant protein yields to levels higher than achieved with wild type expression (120%), reaching over 500 µg/mL of translated protein (comprising 25 µg in 50 µL of reaction mixture). To our knowledge, these results are the highest obtained for both in vivo and in vitro systems. We also demonstrated that efficiency of nonsense suppression depends greatly on the nucleotide following the stop codon. Insights gained in this thorough analysis could prove useful for augmenting in vivo expression levels as well.

Identification of genes influencing synthetic lethality of genetic and epigenetic alterations in translation termination factors in yeast

117 Translation termination in eukaryotic cells is deter mined by proteins Sup35 (eRF3) and Sup45 (eRF1) [1], which interact with a large number of partners [2]. In yeast Saccharomyces cerevisiae, protein Sup35 can form an aggregating epigenetically inherited con former (prion) [PSI+] [3]. This prion is carried through the cytoplasm and causes disturbances in translation termination, which are phenotypically identified as the dominant omnipotent nonsense sup pression. [PSI+] variants with different properties (nonsense suppression efficiency and transmission stability in mitosis) can be obtained in the same yeast strain. The presence of prion [PSI+] leads to lethality in the haploid yeast strain carrying mutations in the gene encoding another termination factor, Sup45 [4]. We have shown that the combination in the diploid strain of some mutant alleles of the SUP45 gene in the heterozygous state with prion [PSI+] entails the death of the hybrid [5]. The synthetic lethality of prion [PSI+] and mutant allele of the sup45 gene depends both on the type of mutant allele and the prion variant. Variant [PSI+], which is a strong suppressor (“strong” [PSI+], or [PSI+]S), causes synthetic lethality with all nonsense mutations and some missense mutations sup45 in the heterozygote. Our data indicate that the lethality of hybrids is correlated with a decreased activity of the Sup45 protein in the cell in case of sup45 mutations. This paper describes a test system that allows identification of proteins that affect the stability of prion [PSI+] and/or the efficiency of translation ter mination by their effect on the synthetic lethality of the prion conformer Sup35 and mutant alleles of SUP45. This test system is suitable to search for pro teins that affect the translation termination efficiency and/or prion maintenance in yeast cells. Gene library screening using this test system allowed us to identify the CUR1 gene, whose influence on another prion, [URE3], was shown earlier but the effect on transla tion termination factors was not known. This test system is based on qualitative assessment of the viability of a diploid strain containing simulta neously (1) prion [PSI+], (2) sup45 mutation in the heterozygote, and (3) test gene(s) in a multicopy plas mid (Fig. 1). Synthetic lethality is manifested as the absence of growth of diploids with genotype SUP45/sup45 [PSI+] (Fig. 2, mutations 101–107, 111, and 116 are incompatible with the “strong” [PSI+]S). If the efficiency of translation termination increases due to the presence of additional copies of the test gene (Х), formation of viable hybrids with genotype +/sup45 [PSI+] [gene Х]n is observed. Conversely, a decrease in the translation termination efficiency in Identification of Genes Influencing Synthetic Lethality of Genetic and Epigenetic Alterations in Translation Termination Factors in Yeast

Protein complex Ppz1p/Hal3p and the efficiency of nonsense suppression in yeasts Saccharomyces cerevisiae

It is known that the efficiency of nonsense suppression in yeasts is controlled both genetically and epigenetically. Since many components of translation machinery are represented by phosphoproteins, the efficiency depends, in particular, on the activity of kinases and phosphatases that include the Ppz1p/Hal3p complex. It contains Ppz1p phosphatase, which is a catalytic subunit, and Hal3p that negatively regulates its function. The aim of this work was to study the mechanisms which relate the activity of Ppz1p/Hal3p complex to nonsense suppression efficiency. In this study, we used a genetic approach implicating the analysis of nonsense suppression phenotype of the strains overexpressing HAL3 or PPZ1 genes and also bearing deletions or mutant alleles of genes, which presumably could participate in the manifestation of these overexpressions. We have shown that Hal3p inhibits not only Ppz1p but also the homologous phosphatase Ppz2p. Our data indicate that Ppz2p is also involved in the control of nonsense suppression efficiency. In the course of search for Ppz1p target protein, it was shown that Ppz1p dephosphorylates at least two proteins involved in translation. Moreover, Ppz1p affects the efficiency of nonsense suppression not only due to its phosphatase activity but also due to another mechanism triggered by its interaction with Hsp70 chaperones.

10.1007/s11177-008-2004-4

The phenomenon of nonsense suppression, which leads to the stop codons reading-through, may be related to disturbances in the operation of various components of the translation apparatus and the proteins interacting with them. The phosphatase Ppzlp is one of the factors affecting the nonsense suppression efficiency in Saccharomyces yeast. In this work, the impact of the overexpression of gene PPZ1 and its mutant allele PPZ1-R451L on the phenotypic expression of various mutant alleles of genes SUP35 and SUP45 or the yeast prion [PSI +] was analyzed. On the basis of the data obtained, a suggestion about the possible role of proteins Sup35p and Sup45p in the processes mediating the influence of gene PPZ1 overexpression on the efficiency of nonsense suppression is made.

Overexpression of gene PPZ1 in the yeast Saccharomyces cerevisiae affects the efficiency of nonsense suppression

The phenomenon of nonsense suppression, which leads to the reading of stop codons as sense codons, may be related to disturbances in the operation of various components of the translation apparatus and the proteins interacting with them. The phosphatase Ppzlp is one of the factors affecting the nonsense suppression efficiency in the saccharomycete yeast. In this work, the impact of the overexpression of gene PPZ1 and its mutant allele PPZ1-R451L on the phenotypic expression of various mutant alleles of genes SUP35 and SUP45 or the yeast prion [PSI+] was analyzed. On the basis of the data obtained, a suggestion about the possible role of proteins Sup35p and Sup45p in the processes mediating the influence of gene PPZ1 overexpression on the efficiency of nonsense suppression is made.

The HAL3‐PPZ1 dependent regulation of nonsense suppression efficiency in yeast and its influence on manifestation of the yeast prion‐like determinant [ISP+

The efficiency of stop codons read-through in yeast is controlled by multiple interactions of genetic and epigenetic factors. In this study, we demonstrate the participation of the Hal3-Ppz1 protein complex in regulation of read-through efficiency and manifestation of non-Mendelian anti-suppressor determinant [ISP(+)]. Over-expression of HAL3 in [ISP(+)] strain causes nonsense suppression, whereas its inactivation displays as anti-suppression of sup35 mutation in [isp(-)] strain. [ISP(+)] strains carrying hal3Delta deletion cannot be cured from [ISP(+)] in the presence of GuHCl. Since Hal3p is a negative regulatory subunit of Ppz1 protein phosphatase, consequences of PPZ1 over-expression and deletion are opposite to those of HAL3. The observed effects are mediated by the catalytic function of Ppz1 and are probably related to the participation of Ppz1 in regulation of eEF1Balpha elongation factor activity. Importantly, [ISP(+)] status of yeast strains is determined by fluctuation in Hal3p level, since [ISP(+)] strains have less Hal3p than their [isp(-)] derivatives obtained by GuHCl treatment. A model considering epigenetic (possibly prion) regulation of Hal3p amount as a mechanism underlying [ISP(+)] status of yeast cell is suggested.

N-terminal extension ofSaccharomyces cerevisiaetranslation termination factor eRF3 influences the suppression efficiency ofsup35mutations

The eukaryotic translation termination factor eRF3 stimulates release of nascent polypeptides from the ribosome in a GTP-dependent manner. In most eukaryotes studied, eRF3 consists of an essential, conserved C-terminal domain and a nonessential, nonconserved N-terminal extension. However, in some species, this extension is required for efficient termination. Our data show that the N-terminal extension of Saccharomyces cerevisiae eRF3 also participates in regulation of termination efficiency, but acts as a negative factor, increasing nonsense suppression efficiency in sup35 mutants containing amino acid substitutions in the C-terminal domain of the protein.

The [ PSI+] prion of yeast: A problem of inheritance

The [ PSI +] prion of the yeast Saccharomyces cerevisiae was first identified by Brian Cox some 40 years ago as a non-Mendelian genetic element that modulated the efficiency of nonsense suppression. Following the suggestion by Reed Wickner in 1994 that such elements might be accounted for by invoking a prion-based model, it was subsequently established that the [ PSI +] determinant was the prion form of the Sup35p protein. In this article, we review how a combination of classical genetic approaches and modern molecular and biochemical methods has provided conclusive evidence of the prion basis of the [ PSI +] determinant. In so doing we have tried to provide a historical context, but also describe the results of more recent experiments aimed at elucidating the mechanism by which the [ PSI +] (and other yeast prions) are efficiently propagated in dividing cells. While understanding of the [ PSI +] prion and its mode of propagation has, and will continue to have, an impact on mammalian prion biology nevertheless the very existence of a protein-based mechanism that can have a beneficial impact on a cell’s fitness provides equally sound justification to fully explore yeast prions.

Effect of temperature and ATP supply on the efficiency of programmed nonsense suppression

Chemical diversity of protein molecules can be expanded through in vitro incorporation of unnatural amino acids in response to a nonsense codon. Chemically misacylated tRNAs are used for tethering unnatural amino acids to a nonsense-mutated target codon (nonsense suppression). In the course of experiments to introduce S-(2-nitrobenzyl)cysteine (NBC) into a targeted location of human erythropoietin, we found that NBC incorporates more efficiently at lower temperatures. In addition, at a fixed reaction temperature, more NBC was incorporated with a reduced supply of ATP. Since the rate of peptide elongation was remarkably higher at the elevated temperature or with enhanced supply of ATP, these results indicate that the efficiency of nonsense suppression is inversely correlated to the peptide elongation rate. Therefore, maximal yield of nonsense-suppressed proteins is obtained at a compromised elongation rate. The present result will offer a primary guideline to optimize the reaction conditions for in vitro production of protein molecules containing unnatural amino acids.

Supporting the structural basis of prion strains: induction and identification of [PSI] variants

The [PSI] genetic element, which enhances the nonsense suppression efficiency in the yeast Saccharomyces cerevisiae, is thought to be amyloid-like aggregates of the Sup35 protein, and to self-propagate by a prion-like mechanism. Analogous to strains of the mammalian prion, variants of [PSI], with different nonsense suppression efficiencies and mitotic stabilities, can be isolated from the same yeast genetic background. In the framework of the “protein-only” hypothesis, variants of prion are assumed to be distinct conformers of the same prion polypeptide. This study aims to provide further support for the structural basis of [PSI] variation. Three variants of [PSI] were induced and distinguished by a panel of 11 single point mutations of the Sup35 protein. The variant phenotypes are intrinsically associated with [PSI] elements, presumably structurally different amyloids, rather than produced from variations in the genetic background. Differential incorporation to [PSI] variants of a Sup35 point mutation as well as N and C-terminally truncated Sup35 fragments is further demonstrated in vivo, suggesting that distinct patches of amino acid residues are involved in the assembly of [PSI] variants. These results establish a method for [PSI] variant-typing and indicate that heritable variations of amyloid structures can be derived from the same polypeptide.

Nonsense-mediated decay mutants do not affect programmed -1 frameshifting.

Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.

The PNM2 mutation in the prion protein domain of SUP35 has distinct effects on different variants of the [PSI+] prion in yeast.

We have previously described different variants of the yeast prion [PSI+] that can be obtained and maintained in the same genetic background. These [PSI+] variants, which differ in the efficiency of nonsense suppression, mitotic stability and the efficiency of curing by GuHCl, may correspond to different [PSI+] prion conformations of Sup35p or to different types of prion aggregates. Here we investigate the effects of overexpressing a mutant allele of SUP35 and find different effects on weak and strong [PSI+] variants: the suppressor phenotype of weak [PSI+] factors is increased, whereas the suppressor effect of strong [PSI+] factors is reduced. The SUP35 mutation used was originally described as a "Psi no more" mutation (PNM2) because it caused loss of [PSI+]. However, none of the [PSI+] variants in the strains used in our study were cured by PNM2. Indeed, when overexpressed, PNM2 induced the de novo appearance of both weak and strong [PSI+] variants with approximately the same efficiency as the overexpressed wild-type SUP35 allele. Our data suggest that the change in the region of oligopeptide repeats in the Sup35p N-terminus due to the PNM2 mutation modifies, but does not impair, the function of the prion domain of Sup35p.

Context Effects on Misreading and Suppression at UAG Codons in Human Cells

The effect of the 3' codon context on the efficiency of nonsense suppression in mammalian tissue culture cells has been tested. Measurements were made following the transfection of cells with a pRSVgal reporter vector that contained the classical Escherichia coli lacZ UAG allele YA559. The position of this mutation was mapped by virtue of its fortuitous creation of a CTAG MaeI restriction enzyme site. Determination of the local DNA sequence revealed a C-->T mutation at codon 600 of the lacZ gene: CAG-->TAG. Site-directed mutagenesis was used to create a series of vectors in which the base 3' to the nonsense codon was either A, C, G, or U. Suppression of the amber-containing reporter was achieved by cotransfection with genes for human tRNA(Ser) or tRNA(Gln) UAG nonsense suppressors and by growth in the translational error-promoting aminoglycoside drug G418. Nonsense suppression was studied in the human cell lines 293 and MRC5V1 and the simian line COS-7. Overall, the rank order for the effect of changes to the base 3' to UAG was C < G = U < A. This study confirms and extends earlier findings that in mammalian cells 3' C supports efficient nonsense suppression while 3' A is unsympathetic for read-through at nonsense codons. The rules for the mammalian codon context effect on nonsense suppression are therefore demonstrably different from those in E. coli.

Isolation and characterization of antisuppressor mutations in Escherichia coli.

Nonsense mutations in lacI have been shown to be useful as indicators of the efficiency of nonsense suppression. From strains containing supE and a lacI nonsense mutation, selection for LacI- mutants has resulted in the isolation of four antisuppressor mutations. Tn10 insertions linked to these mutations were isolated and used to group the four mutations into three loci. The asuA1 and asuA2 mutations are linked to trp, reduce suppression by supE approximately twofold, and affect a variety of suppressors. The asuB3 mutation was mapped by P1 cotransduction to rpsL but does not confer resistance to streptomycin. The asuC4 mutation reduced suppression by supE by 95% and was shown biochemically to result in the loss of two pseudouridine modifications from the 3' side of the anticodon stem and loop of tRNA2Gln. This mutation is linked to purF, suggesting that it is a new allele of hisT.

An homologous in vitro assay for yeast nonsense suppressors.

A cell-free translation system, from the yeast Saccharomyces cerevisiae, has been used to develop an in vitro assay for yeast UGA, ochre and amber suppressors. Amber suppression was assayed by read-through of the brome mosaic virus coat protein cistron UAG terminator. UGA suppression was assayed by read-through of the rabbit beta-globin UGA terminator and ochre suppression by read-through of the rabbit alpha-globin mRNA UAA terminator. Ochre suppression was increased 3-fold when the globin mRNA was heat denatured prior to translation; this was due to an increase in the synthesis of alpha-globin relative to beta-globin. Amber suppression was more efficient in vitro (46%) than ochre suppression (14%). UGA suppression was also highly efficient in vitro, reaching almost 100% using a purified UGA suppressor tRNA from Schizosaccharomyces pombe. Unfractionated yeast tRNA, from a sup+ strain, contained a tRNA species able to suppress UGA termination codons in vitro, but no tRNA species able to suppress either UAA or UAG was found. This homologous in vitro assay for yeast nonsense suppressors will allow, for the first time, an approach to the biochemical analysis of yeast mutants that modify the efficiency of nonsense suppression in vivo.

Ribosomal proteins of yeast strains carrying mutations which affect the efficiency of nonsense suppression

We have examined the ribosomal proteins of strains of Saccharomyces cerevisiae which differ in the efficiency with which ochre nonsense mutations are suppressed. The strains in which ochre suppression is poor were [psi]- or carried antisuppressor mutations; those in which suppression was highly efficient were [psi]+ or carried allosuppressor mutations. The ribosomal proteins of these strains, as judged by two-dimensional polyacrylamide gel electrophoresis, were indistinguishable from those of wild-type.