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1.  Statistical Analysis of Readthrough Levels for Nonsense Mutations in Mammalian Cells Reveals a Major Determinant of Response to Gentamicin 
PLoS Genetics  2012;8(3):e1002608.
The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the −1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable.
Author Summary
Nonsense mutations are single-nucleotide variations within the coding sequence of a gene that result in a premature termination codon. The presence of such mutations leads to the synthesis of a truncated protein unable to fulfill its normal function. Over the last ten years, treatment strategies have emerged based on the use of molecules, such as aminoglycoside antibiotics (gentamicin) that facilitate the readthrough of premature termination codons, thus restoring the synthesis of a full-length protein. Such strategies have been tested for various genetic diseases, including Duchenne muscular dystrophy and cystic fibrosis. The readthrough level depends on the nature of the stop codon and the surrounding nucleotide context, but little was known of the rules governing readthrough level and response to aminoglycosides. In this study, we use a large set of nonsense mutations for an in-depth statistical analysis designed to decipher the element of the nucleotide context responsible for modulating readthrough levels. We analyse the impact of the six nucleotides upstream and downstream from the stop codon. We demonstrate that the presence of a uracil residue immediately upstream the stop codon is associated with a stronger response to gentamicin treatment than the presence of any of the other three nucleotides.
doi:10.1371/journal.pgen.1002608
PMCID: PMC3315467  PMID: 22479203
2.  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.
ABSTRACT
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.
doi:10.1128/JVI.00898-14
PMCID: PMC4178896  PMID: 24991001
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.
doi:10.1093/nar/gkh791
PMCID: PMC516063  PMID: 15326224
4.  Genetic Basis of Hidden Phenotypic Variation Revealed by Increased Translational Readthrough in Yeast 
PLoS Genetics  2012;8(3):e1002546.
Eukaryotic release factors 1 and 3, encoded by SUP45 and SUP35, respectively, in Saccharomyces cerevisiae, are required for translation termination. Recent studies have shown that, besides these two key factors, several genetic and epigenetic mechanisms modulate the efficiency of translation termination. These mechanisms, through modifying translation termination fidelity, were shown to affect various cellular processes, such as mRNA degradation, and in some cases could confer a beneficial phenotype to the cell. The most studied example of such a mechanism is [PSI+], the prion conformation of Sup35p, which can have pleiotropic effects on growth that vary among different yeast strains. However, genetic loci underlying such readthrough-dependent, background-specific phenotypes have yet to be identified. Here, we used sup35C653R, a partial loss-of-function allele of the SUP35 previously shown to increase readthrough of stop codons and recapitulate some [PSI+]-dependent phenotypes, to study the genetic basis of phenotypes revealed by increased translational readthrough in two divergent yeast strains: BY4724 (a laboratory strain) and RM11_1a (a wine strain). We first identified growth conditions in which increased readthrough of stop codons by sup35C653R resulted in different growth responses between these two strains. We then used a recently developed linkage mapping technique, extreme QTL mapping (X-QTL), to identify readthrough-dependent loci for the observed growth differences. We further showed that variation in SKY1, an SR protein kinase, underlies a readthrough-dependent locus observed for growth on diamide and hydrogen peroxide. We found that the allelic state of SKY1 interacts with readthrough level and the genetic background to determine growth rate in these two conditions.
Author Summary
Proper termination is an important step in a successful mRNA translation event. Many factors, employing genetic and epigenetic mechanisms, are involved in modifying translation termination efficiency in the budding yeast, Saccharomyces cerevisiae. [PSI+], the prion conformation of Sup35p, one of the translation termination factors in yeast, provides an example of such mechanisms. [PSI+] increases readthrough of stop codons. This has the potential to unveil hidden genetic variation that may enhance growth in some yeast strains in certain environments. The specific details of readthrough-dependent phenotypes, however, have remained poorly understood. Here, we used a partial loss-of-function allele of SUP35, which increases readthrough of stop codons, and a recently developed linkage mapping technique, X-QTL, to map loci underlying readthrough-dependent growth phenotypes in two divergent yeast strains, BY (a laboratory strain) and RM (a wine strain). We found that readthrough-dependent growth phenotypes are often complex, with multiple loci influencing growth. We also showed that variants in the gene SKY1 underlie one of the loci detected for readthrough-dependent growth phenotypes in the presence of two chemicals that induce oxidative stress.
doi:10.1371/journal.pgen.1002546
PMCID: PMC3291563  PMID: 22396662
5.  Genome-wide prediction of stop codon readthrough during translation in the yeast Saccharomyces cerevisiae 
Nucleic Acids Research  2004;32(22):6605-6616.
In-frame stop codons normally signal termination during mRNA translation, but they can be read as ‘sense’ (readthrough) depending on their context, comprising the 6 nt preceding and following the stop codon. To identify novel contexts directing readthrough, under-represented 5′ and 3′ stop codon contexts from Saccharomyces cerevisiae were identified by genome-wide survey in silico. In contrast with the nucleotide bias 3′ of the stop codon, codon bias in the two codon positions 5′ of the termination codon showed no correlation with known effects on stop codon readthrough. However, individually, poor 5′ and 3′ context elements were equally as effective in promoting stop codon readthrough in vivo, readthrough which in both cases responded identically to changes in release factor concentration. A novel method analysing specific nucleotide combinations in the 3′ context region revealed positions +1,2,3,5 and +1,2,3,6 after the stop codon were most predictive of termination efficiency. Downstream of yeast open reading frames (ORFs), further in-frame stop codons were significantly over-represented at the +1, +2 and +3 codon positions after the ORF, acting to limit readthrough. Thus selection against stop codon readthrough is a dominant force acting on 3′, but not on 5′, nucleotides, with detectable selection on nucleotides as far downstream as +6 nucleotides. The approaches described can be employed to define potential readthrough contexts for any genome.
doi:10.1093/nar/gkh1004
PMCID: PMC545446  PMID: 15602002
6.  Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster 
eLife  2013;2:e01179.
Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution.
DOI: http://dx.doi.org/10.7554/eLife.01179.001
eLife digest
For a gene to give rise to a protein, its DNA is first used as a template to produce a messenger RNA molecule. Each group of three nucleotides within the messenger RNA encodes an amino acid, and structures called ribosomes assemble the protein by joining together amino acids in the correct order. The nucleotide triplets are called codons, and some are known as stop codons because they typically instruct the ribosome to stop adding amino acids.
Sometimes ribosomes interpret stop codons as amino acid insertion signals, giving rise to an extended protein with a modified structure or function. This phenomenon is known as stop codon readthrough, and is required for many viruses to complete their reproductive cycles. However, much less is known about stop codon readthrough in other organisms.
Now, Dunn et al. have used a technique called ribosome profiling to analyze stop codon readthrough across the entire genome of the fruit fly Drosophila melanogaster. An enzyme was used to fragment messenger RNA, and those fragments that were specifically engaged by ribosomes—and thus likely to encode protein—were sequenced. Stop codon readthrough occurred much more often than had been expected based on previous studies. Indeed, computational analysis strongly suggests that evolution has favored this process for certain fruit fly genes. Moreover, stop codon readthrough was also observed in yeast and human cells, suggesting that it is important in many organisms, not just the fruit fly.
Stop codon readthrough thus provides a novel way for organisms to tune the expression levels and functions of their genes, both throughout the lifetime of an individual, and the evolution of a species.
DOI: http://dx.doi.org/10.7554/eLife.01179.002
doi:10.7554/eLife.01179
PMCID: PMC3840789  PMID: 24302569
ribosome; translation; readthrough; stop codon; evolution; ribosome profiling; D. melanogaster; Human; S. cerevisiae
7.  Ribosomal Readthrough at a Short UGA Stop Codon Context Triggers Dual Localization of Metabolic Enzymes in Fungi and Animals 
PLoS Genetics  2014;10(10):e1004685.
Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated.
Author Summary
Eukaryotic organisms use various strategies to generate protein isoforms with different function or intracellular localization from a single gene. While differential splicing of mRNA is the most common mechanism to expand the number of encoded proteins, translational readthrough of stop codons is an alternative strategy to create protein variants with C-terminally extended proteins. Recently, it has been shown that fungi use both alternative splicing and translational readthrough to specify peroxisomal isoforms of glycolytic enzymes. Here we show that stop codon readthrough is also used in the animal kingdom to target important metabolic enzymes to peroxisomes. Interestingly, several of these enzymes have a function in peroxisomal redox homeostasis and energy metabolism. It has been described that termination fidelity is modulated by oxidation of specific ribosomal proteins. This suggests that dual targeting via translational readthrough allows adaptation of peroxisomal metabolism to the oxidative status of the cell.
doi:10.1371/journal.pgen.1004685
PMCID: PMC4207609  PMID: 25340584
8.  Functional Analysis of the Interplay between Translation Termination, Selenocysteine Codon Context, and Selenocysteine Insertion Sequence-binding Protein 2* 
A selenocysteine insertion sequence (SECIS) element in the 3′-untranslated region and an in-frame UGA codon are the requisite cis-acting elements for the incorporation of selenocysteine into selenoproteins. Equally important are the trans-acting factors SBP2, Sec-tRNA[Ser]Sec, and eEFSec. Multiple in-frame UGAs and two SECIS elements make the mRNA encoding selenoprotein P (Sel P) unique. To study the role of codon context in determining the efficiency of UGA readthrough at each of the 10 rat Sel P Sec codons, we individually cloned 27-nucleotide-long fragments representing each UGA codon context into a luciferase reporter construct harboring both Sel P SECIS elements. Significant differences, spanning an 8-fold range of UGA readthrough efficiency, were observed, but these differences were dramatically reduced in the presence of excess SBP2. Mutational analysis of the “fourth base” of contexts 1 and 5 revealed that only the latter followed the established rules for hierarchy of translation termination. In addition, mutations in either or both of the Sel P SECIS elements resulted in differential effects on UGA readthrough. Interestingly, even when both SECIS elements harbored a mutation of the core region required for Sec incorporation, context 5 retained a significantly higher level of readthrough than context 1. We also show that SBP2-dependent Sec incorporation is able to repress G418-induced UGA readthrough as well as eRF1-induced stimulation of termination. We conclude that a large codon context forms a cis-element that works together with Sec incorporation factors to determine readthrough efficiency.
doi:10.1074/jbc.M707061200
PMCID: PMC2820277  PMID: 17954931
9.  Pseudouridine in the anticodon G psi A of plant cytoplasmic tRNA(Tyr) is required for UAG and UAA suppression in the TMV-specific context. 
Nucleic Acids Research  1992;20(22):5911-5918.
We have previously isolated and sequenced Nicotiana cytoplasmic tRNA(Tyr) with G psi A anticodon which promotes readthrough over the leaky UAG termination codon at the end of the 126 K cistron of tobacco mosaic virus RNA and we have demonstrated that tRNA(Tyr) with Q psi A anticodon is no UAG suppressor. Here we show that the nucleotide in the middle of the anticodon (i.e., psi 35) also contributes to the suppressor efficiency displayed by cytoplasmic tRNA(Tyr). A tRNA(Tyr) with GUA anticodon was synthesized in vitro using T7 RNA polymerase transcription. This tRNA(Tyr) was unable to suppress the UAG codon, indicating that nucleotide modifications in the anticodon of tRNA(Tyr) have either stimulating (i.e., psi 35) or inhibitory (i.e., Q34) effects on suppressor activity. Furthermore, we have shown that the UAA but not the UGA stop codon is also efficiently recognized by tobacco tRNA(G psi ATyr), if placed in the TMV context. Hence this is the first naturally occurring tRNA for which UAA suppressor activity has been demonstrated. In order to study the influence of neighbouring nucleotides on the readthrough capacity of tRNA(Tyr), we have established a system, in which part of the sequence around the leaky UAG codon of TMV RNA was inserted into a zein pseudogene which naturally harbours an UAG codon in the middle of the gene. The construct was cloned into the vector pSP65 and in vitro transcripts, generated by SP6 RNA polymerase, were translated in a wheat germ extract depleted of endogenous mRNAs and tRNAs. A number of mutations in the codons flanking the UAG were introduced by site-directed mutagenesis. It was found that changes at specific positions of the two downstream codons completely abolished the readthrough over the UAG by Nicotiana tRNA(Tyr), indicating that this tRNA needs a very specific codon context for its suppressor activity.
Images
PMCID: PMC334454  PMID: 1461724
10.  The signal for translational readthrough of a UGA codon in Sindbis virus RNA involves a single cytidine residue immediately downstream of the termination codon. 
Journal of Virology  1993;67(8):5062-5067.
The nucleotide sequences surrounding termination codons influence the efficiency of translational readthrough. In this report, we examined the sequence requirement for efficient readthrough of the UGA codon in the Sindbis virus genomic RNA which regulates production of the putative viral RNA polymerase, nsP4. The UGA codon and its neighboring nucleotide sequences were subcloned into a heterologous coding context, and readthrough efficiency was measured by cell-free translation of RNA transcripts in rabbit reticulocyte lysates. The CUA codon immediately downstream of the UGA codon was found to be sufficient for efficient translational readthrough. Further mutagenesis of residues in the CUA triplet demonstrated that mutations at the second or third residues following the UGA codon (U and A, respectively) had little effect on readthrough efficiency. In contrast, replacement of the cytidine residue immediately downstream of the UGA codon with any of the other three nucleotides (U, A, or G) dramatically reduced the readthrough efficiency from approximately 10% to less than 1%. These results show that a simple sequence context can allow efficient readthrough of UGA codons in a mammalian translation system. Interestingly, compilation studies of nucleotide sequences surrounding eukaryotic termination codons indicate a strong bias against cytidine residues immediately 3' to UGA termination codons. Taken together with our results, this bias may reflect a selective pressure for efficient translation termination for most eukaryotic gene products.
Images
PMCID: PMC237898  PMID: 8331741
11.  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
12.  Variants in SUP45 and TRM10 Underlie Natural Variation in Translation Termination Efficiency in Saccharomyces cerevisiae 
PLoS Genetics  2011;7(7):e1002211.
Translation termination is a highly controlled process in the cell. In Saccharomyces cerevisiae, various regulatory factors employ genetic and epigenetic mechanisms to control this process. We used a quantitative dual luciferase reporter assay to demonstrate a difference in translation termination efficiency between two different yeast strains, BY4724 and RM11-1a. We then used a recently developed linkage mapping technique, extreme QTL mapping (X-QTL), to show that this difference is largely explained by a coding polymorphism in TRM10 (which encodes a tRNA–methylating enzyme) and a regulatory polymorphism in SUP45 (which encodes one of the yeast translation termination factors). BY and RM carry variants of TRM10 and SUP45 with opposite effects on translation termination efficiency. These variants are common among 63 diverse S. cerevisiae strains and are in strong linkage disequilibrium with each other. This observation suggests that selection may have favored allelic combinations of the two genes that maintain an intermediate level of translation termination efficiency. Our results also provide genetic evidence for a new role of Trm10p in translation termination efficiency.
Author Summary
Translation, the process of protein synthesis from messenger RNA (mRNA), cannot be successfully completed without proper termination. The ends of the mRNA coding regions are marked by one of the three stop codons, which are recognized by termination factors rather than by the transfer RNAs (tRNAs) that match amino acids to the corresponding codons. Like most biological processes, translation termination is not perfect. Occasionally, tRNAs bind to stop codons, resulting in polypeptides with additional amino acids beyond the normal stop position—a phenomenon known as readthrough. Perturbations that affect the balance between termination factors and tRNAs will change readthrough. Here we demonstrate the effect of two perturbations on translation termination efficiency in the context of natural genetic variation. We show that a difference in readthrough between a laboratory and a vineyard strain of yeast is largely due to two genetic variants. One variant affects the expression level of a key translation termination factor; the other modifies the activity of a tRNA–methylating enzyme. We also show that natural selection has favored an intermediate level of readthrough.
doi:10.1371/journal.pgen.1002211
PMCID: PMC3145625  PMID: 21829385
13.  Versatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plants 
PLoS ONE  2009;4(10):e7354.
Background
Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA) in an mRNA. However, many viruses (and some cellular mRNAs) contain “stop” codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a “readthrough”, or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context.
Methodology
We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP.
Conclusions
We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a ‘leaky’ UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV) readthrough context.
doi:10.1371/journal.pone.0007354
PMCID: PMC2754532  PMID: 19816579
14.  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
15.  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.
doi:10.1128/MCB.25.14.5801-5811.2005
PMCID: PMC1168810  PMID: 15987998
16.  5' contexts of Escherichia coli and human termination codons are similar. 
Nucleic Acids Research  1995;23(22):4712-4716.
The nearest 5' context of 2559 human stop codons was analysed in comparison with the same context of stop-like codons (UGG, UGC, UGU, CGA for UGA; CAA, UAU, UAC for UAA; and UGG, UAU, UAC, CAG for UAG). The non-random distribution of some nucleotides upstream of the stop codons was observed. For instance, uridine is over-represented in position -3 upstream of UAG. Several codons were shown to be over-represented immediately upstream of the stop codons: UUU(Phe), AGC(Ser), and the Lys and Ala codon families before UGA; AAG(Lys), GCG(Ala), and the Ser and Leu codon families before UAA; and UCA(Ser), AUG(Met), and the Phe codon family before UAG. In contrast, the Thr and Gly codon families were under-represented before UGA, while ACC(Thr) and the Gly codon family were under-represented before UAG and UAA respectively. In an earlier study, uridine was shown to be over-represented in position -3 before UGA in Escherichia coli [Arkov,A.L., Korolev,S.V. and Kisselev,L.L. (1993) Nucleic Acids Res., 21,2891-2897]. In that study, the codons for Lys, Phe and Ser were shown to be over-represented immediately upstream of E. coli stop codons. Consequently, E. coli and human termination codons have similar 5' contexts. The present study suggests that the 5' context of stop codons may modulate the efficiency of peptide chain termination and (or) stop codon readthrough in higher eukaryotes, and that the mechanisms of such a modulation in prokaryotes and higher eukaryotes may be very similar.
PMCID: PMC307448  PMID: 8524665
17.  Control of gag-pol gene expression in the Candida albicans retrotransposon Tca2 
Background
In the C. albicans retrotransposon Tca2, the gag and pol ORFs are separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However, in other retroelements, translational readthrough of the gag stop codon can be directed by its flanking sequence, including a 3' pseudoknot.
Results
The hypothesis was tested that in Tca2, gag stop codon flanking sequences direct translational readthrough and synthesis of a gag-pol fusion protein. Sequence from the Tca2 gag-UGA-pol junction (300 nt) was inserted between fused lacZ and luciferase (luc) genes in a Saccharomyces cerevisiae dual reporter construct. Although downstream of UGA, luc was expressed, but its expression was unaffected by inserting additional stop codons at the 3' end of lacZ. Luc expression was instead being driven by a previously unknown minor promoter activity within the gag-pol junction region. Evidence together indicated that junction sequence alone cannot direct UGA readthrough. Using reporter genes in C. albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR) promoter were compared. Of the two promoters, only the LTR promoter was induced by heat-shock, which also triggers retrotransposition. Tca2 pol protein, epitope-tagged in C. albicans to allow detection, was also heat-shock induced, indicating that pol proteins were expressed from a gag-UGA-pol RNA.
Conclusion
This is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass. However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S. cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2.
doi:10.1186/1471-2199-8-94
PMCID: PMC2194720  PMID: 17961216
18.  New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae 
Nucleic Acids Research  2014;42(15):10061-10072.
Stop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5′ nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNAGln were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.
doi:10.1093/nar/gku663
PMCID: PMC4150775  PMID: 25056309
19.  Evidence of efficient stop codon readthrough in four mammalian genes 
Nucleic Acids Research  2014;42(14):8928-8938.
Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes. Here we use a modified targeted bioinformatic approach to identify a further three mammalian readthrough candidates. All seven genes were tested experimentally using reporter constructs transfected into HEK-293T cells. Four displayed efficient stop codon readthrough, and these have UGA immediately followed by CUAG. Comparative genomic analysis revealed that in the four readthrough candidates containing UGA-CUAG, this motif is conserved not only in mammals but throughout vertebrates with the first six of the seven nucleotides being universally conserved. The importance of the CUAG motif was confirmed using a systematic mutagenesis approach. One gene, OPRL1, encoding an opiate receptor, displayed extremely efficient levels of readthrough (∼31%) in HEK-293T cells. Signals both 5′ and 3′ of the OPRL1 stop codon contribute to this high level of readthrough. The sequence UGA-CUA alone can support 1.5% readthrough, underlying its importance.
doi:10.1093/nar/gku608
PMCID: PMC4132726  PMID: 25013167
20.  Readthrough of nonsense mutation W822X in the SCN5A gene can effectively restore expression of cardiac Na+ channels 
Cardiovascular Research  2009;83(3):473-480.
Aims
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.
Conclusion
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.
doi:10.1093/cvr/cvp116
PMCID: PMC2709462  PMID: 19377070
Sodium channels; Aminoglycosides; Eukaryotic release factors; siRNA; Nonsense mutation
21.  Multifaceted Regulation of Translational Readthrough by RNA Replication Elements in a Tombusvirus 
PLoS Pathogens  2011;7(12):e1002423.
Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp) in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i) providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii) mediating cis-preferential replication of viral genomes, and (iii) coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family.
Author Summary
Viruses use many different strategies to produce their proteins and some viral proteins are made with terminal extensions that confer unique properties. The polymerase that replicates the RNA genomes of tombusviruses is an extended version of another viral protein and is generated by a process called translational readthrough. We have determined the regulatory mechanism that modulates the production of this viral polymerase. Our results show that control of the readthrough process is complex and involves both local structures and long-range interactions within the viral genome. This system is also integrated with viral RNA replication elements and this allows the virus to coordinate polymerase production with genome replication. This regulatory scheme appears to represent a common tactic used by a variety of viruses.
doi:10.1371/journal.ppat.1002423
PMCID: PMC3234231  PMID: 22174683
22.  Stops making sense: translational trade-offs and stop codon reassignment 
Background
Efficient gene expression involves a trade-off between (i) premature termination of protein synthesis; and (ii) readthrough, where the ribosome fails to dissociate at the terminal stop. Sense codons that are similar in sequence to stop codons are more susceptible to nonsense mutation, and are also likely to be more susceptible to transcriptional or translational errors causing premature termination. We therefore expect this trade-off to be influenced by the number of stop codons in the genetic code. Although genetic codes are highly constrained, stop codon number appears to be their most volatile feature.
Results
In the human genome, codons readily mutable to stops are underrepresented in coding sequences. We construct a simple mathematical model based on the relative likelihoods of premature termination and readthrough. When readthrough occurs, the resultant protein has a tail of amino acid residues incorrectly added to the C-terminus. Our results depend strongly on the number of stop codons in the genetic code. When the code has more stop codons, premature termination is relatively more likely, particularly for longer genes. When the code has fewer stop codons, the length of the tail added by readthrough will, on average, be longer, and thus more deleterious. Comparative analysis of taxa with a range of stop codon numbers suggests that genomes whose code includes more stop codons have shorter coding sequences.
Conclusions
We suggest that the differing trade-offs presented by alternative genetic codes may result in differences in genome structure. More speculatively, multiple stop codons may mitigate readthrough, counteracting the disadvantage of a higher rate of nonsense mutation. This could help explain the puzzling overrepresentation of stop codons in the canonical genetic code and most variants.
doi:10.1186/1471-2148-11-227
PMCID: PMC3161013  PMID: 21801361
23.  Efficient Multisite Unnatural Amino Acid Incorporation in Mammalian Cells via Optimized Pyrrolysyl tRNA Synthetase/tRNA Expression and Engineered eRF1 
Journal of the American Chemical Society  2014;136(44):15577-15583.
The efficient, site-specific introduction of unnatural amino acids into proteins in mammalian cells is an outstanding challenge in realizing the potential of genetic code expansion approaches. Addressing this challenge will allow the synthesis of modified recombinant proteins and augment emerging strategies that introduce new chemical functionalities into proteins to control and image their function with high spatial and temporal precision in cells. The efficiency of unnatural amino acid incorporation in response to the amber stop codon (UAG) in mammalian cells is commonly considered to be low. Here we demonstrate that tRNA levels can be limiting for unnatural amino acid incorporation efficiency, and we develop an optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system, with optimized tRNA expression for mammalian cells. In addition, we engineer eRF1, that normally terminates translation on all three stop codons, to provide a substantial increase in unnatural amino acid incorporation in response to the UAG codon without increasing readthrough of other stop codons. By combining the optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system and an engineered eRF1, we increase the yield of protein bearing unnatural amino acids at a single site 17- to 20-fold. Using the optimized system, we produce proteins containing unnatural amino acids with comparable yields to a protein produced from a gene that does not contain a UAG stop codon. Moreover, the optimized system increases the yield of protein, incorporating an unnatural amino acid at three sites, from unmeasurably low levels up to 43% of a no amber stop control. Our approach may enable the efficient production of site-specifically modified therapeutic proteins, and the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function.
doi:10.1021/ja5069728
PMCID: PMC4333590  PMID: 25350841
24.  N-terminal region of Saccharomyces cerevisiae eRF3 is essential for the functioning of the eRF1/eRF3 complex beyond translation termination 
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2199-7-34
PMCID: PMC1617110  PMID: 17034622
25.  Effects of an Opal Termination Codon Preceding the nsP4 Gene Sequence in the O'Nyong-Nyong Virus Genome on Anopheles gambiae Infectivity 
Journal of Virology  2006;80(10):4992-4997.
The genomic RNA of an alphavirus encodes four different nonstructural proteins, nsP1, nsP2, nsP3, and nsP4. The polyprotein P123 is produced when translation terminates at an opal termination codon between nsP3 and nsP4. The polyprotein P1234 is produced when translational readthrough occurs or when the opal termination codon has been replaced by a sense codon in the alphavirus genome. Evolutionary pressures appear to have maintained genomic sequences encoding both a stop codon (opal) and an open reading frame (arginine) as a general feature of the O'nyong-nyong virus (ONNV) genome, indicating that both are required at some point. Alternate replication of ONNVs in both vertebrate and invertebrate hosts may determine predominance of a particular codon at this locus in the viral quasispecies. However, no systematic study has previously tested this hypothesis in whole animals. We report here the results of the first study to investigate in a natural mosquito host the functional significance of the opal stop codon in an alphavirus genome. We used a full-length cDNA clone of ONNV to construct a series of mutants in which the arginine between nsP3 and nsP4 was replaced with an opal, ochre, or amber stop codon. The presence of an opal stop codon upstream of nsP4 nearly doubled (75.5%) the infectivity of ONNV over that of virus possessing a codon for the amino acid arginine at the corresponding position (39.8%). Although the frequency with which the opal virus disseminated from the mosquito midgut did not differ significantly from that of the arginine virus on days 8 and 10, dissemination did began earlier in mosquitoes infected with the opal virus. Although a clear fitness advantage is provided to ONNV by the presence of an opal codon between nsP3 and nsP4 in Anopheles gambiae, sequence analysis of ONNV RNA extracted from mosquito bodies and heads indicated codon usage at this position corresponded with that of the virus administered in the blood meal. These results suggest that while selection of ONNV variants is occurring, de novo mutation at the position between nsP3 and nsP4 does not readily occur in the mosquito. Taken together, these results suggest that the primary fitness advantage provided to ONNV by the presence of an opal codon between nsP3 and nsP4 is related to mosquito infectivity.
doi:10.1128/JVI.80.10.4992-4997.2006
PMCID: PMC1472075  PMID: 16641290

Results 1-25 (1426703)