Readthrough Efficiency Research Articles

Predicting the efficiency of UAG translational stop signal through studies of physicochemical properties of its composite mono- and dinucleotides

In this study, we explored the problem of predicting the UAG stop-codon read-through efficiency. The reported nucleotide sequences were first converted into physicochemical property vectors before being presented to a machine learning algorithm. Two sets of physicochemical properties were applied: one for mononucleosides (in terms of steric bulk, hydrophobicity and electronics) and another for dinucleotides. To the best of our knowledge, this is the first report of how dinucleotides are converted into principle components derived from NMR chemical shift data. A few efficiency prediction models were then derived and a comparison between mononucleoside and dinucleotide-based models was shown. In the derived models, the coefficients of these property based predictors lend themselves to bio-physical interpretations, an advantage which is demonstrated in this study via a prediction model based on the steric bulk factor. Although it is quite simple, the steric bulk factor model explained well the effect of sequence variations surrounding the amber stop codon and the tRNA bearing UCCU anticodon. We further proposed new alternatives at position −1 and +4 of a UAG stop codon sequence to enhance the readthrough efficiency. This research may contribute to a better understanding of the readthrough mechanisms and may also help to study the normal translation termination process.

Computational identification and sequence analysis of stop codon readthrough genes in Oryza sativa

Using an approach based on the Readthrough Candidate Extraction System (RCES), we extracted 111 candidates from 9620 gene sequences of rice. The results of homology search and sequence analysis demonstrated that these candidates included actual readthrough genes that would be important for further investigating the mechanism of translation termination regulated by readthrough event, and could also give some useful clues for functional genome annotation. Between the candidates and non-candidates of gene sequences in rice, there exist significant base biases at the positions surrounding the stop codons. These positions, especially both −1 and +4, are referred to as part of an extended stop signal. In candidates, G at position −1, and G or C at position +4 are much more favored than that in non-candidates. Both stop sequence patterns, GUAGC and GUGAG, might drive high readthrough efficiency in rice. Secondary structure analysis revealed that the −1 and +1 amino acids around the first stop codon of candidates have a strong bias toward arginine, particularly the +1 position (20.7%), which indicated that the amino acids at the readthrough region being frequently located in the hydrophilic region of β-turn might be a determinant for efficient translation termination or not.

Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment.

The suppression levels induced by gentamicin on premature stop codons, caused by primary nonsense mutations found in muscular dystrophy patients, were assessed using a very sensitive dual reporter gene assay. Results show that: (i) the effect of gentamicin on readthrough is similar in cultured cells and in vivo in murine skeletal muscle; (ii) a wide variability of readthrough efficiency is obtained, depending on the mutation tested; (iii) due to the complexity of readthrough regulation, efficiency cannot be predicted by the nucleotide context of the stop codon; (iv) only a minority of premature stop codons found in patients show a significant level of readthrough, and would thus be amenable to this pharmacological treatment, given our present understanding of the problem. These results probably provide an explanation for the relative failure of clinical trials reported to date using gentamicin to treat diseases due to premature stop codons, and emphasize that preliminary assays in cell culture provide valuable information concerning the potential efficiency of pharmacological treatments.

The major 5' determinant in stop codon read-through involves two adjacent adenines.

The aim of this approach was to identify the major determinants, located at the 5' end of the stop codon, that modulate translational read-through in Saccharomyces cerevisiae. We developed a library of oligonucleotides degenerate at the six positions immediately upstream of the termination codon, cloned in the ADE2 reporter gene. Variations at these positions modulated translational read-through efficiency approximately 16-fold. The major effect was imposed by the two nucleotides immediately upstream of the stop codon. We showed that this effect was neither mediated by the last amino acid residues present in the polypeptide chain nor by the tRNA present in the ribosomal P site. We propose that the mRNA structure, depending on the nucleotides in the P site, is the main 5' determinant of read-through efficiency.

Lack of Pseudouridine 38/39 in the Anticodon Arm of Yeast Cytoplasmic tRNA Decreases in Vivo Recoding Efficiency

Many different modified nucleotides are found in naturally occurring tRNA, especially in the anticodon region. Their importance for the efficiency of the translational process begins to be well documented. Here we have analyzed the in vivo effect of deleting genes coding for yeast tRNA-modifying enzymes, namely Pus1p, Pus3p, Pus4p, or Trm4p, on termination readthrough and +1 frameshift events. To this end, we have transformed each of the yeast deletion strains with a lacZ-luc dual-reporter vector harboring selected programmed recoding sites. We have found that only deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In this mutant, we have observed a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA because the inactive mutant protein Pus3[D151A]p did not restore the level of natural readthrough. Our results also show that absence of pseudouridine 39 in the slippery tRNA(UAG)(Leu) reduces +1 frameshift efficiency. Therefore, the presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast.

A dynamic competition between release factor 2 and the tRNASec decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H

Factors affecting competition between termination and elongation in vivo during translation of the fdhF selenocysteine recoding site (UGA) were studied with wild-type and modified fdhF sequences. Altering sequences surrounding the recoding site UGA without affecting RNA secondary structure indicated that the kinetics of stop signal decoding have a significant influence on selenocysteine incorporation efficiency. The UGA in the wild-type fdhF sequence remains 'visible' to the factor and forms a site-directed cross-link when mRNA stem-loop secondary structure is absent, but not when it is present. The timing of the secondary structure unfolding during translation may be a critical feature of competition between release factor 2 and tRNA(Sec) for decoding UGA. Increasing the cellular concentration of either of these decoding molecules for termination or selenocysteine incorporation showed that they were able to compete for UGA by a kinetic competition that is dynamic and dependent on the Escherichia coli growth rate. The tRNA(Sec)-mediated decoding can compete more effectively for the UGA recoding site at lower growth rates, consistent with anaerobic induction of fdhF expression.

Impact of the six nucleotides downstream of the stop codon on translation termination.

The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial-based strategy to identify poor 3' termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence -CA(A/G)N(U/C/G)A-, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3' stop codon context on translation termination.

A novel stop codon readthrough mechanism produces functional Headcase protein in Drosophila trachea.

Translational regulation provides an efficient means to control the localization and production of proteins. The headcase (hdc) mRNA in Drosophila generates two overlapping proteins as a result of translational readthrough of an internal UAA stop codon. This readthrough event is necessary for the function of hdc as a branching inhibitor during tracheal development. By ectopic expression of different Hdc proteins in the trachea, we show that the long Hdc form alone, can function as a potent branching inhibitor whose activity is proportional to its amount. The suppression of termination in the hdc mRNA is not stop-codon dependent, suggesting that the readthrough does not involve codon specific suppressors. We have identified an 80 nucleotide sequence immediately downstream of the UAA, which is necessary and sufficient to confer termination readthrough in a heterologous mRNA. We present a novel mechanism of eukaryotic translational termination suppression that may regulate the amount of functional Hdc.

UAG readthrough in mammalian cells: Effect of upstream and downstream stop codon contexts reveal different signals

BackgroundTranslation termination is mediated through an interaction between the release factors eRF1 and eRF3 and the stop codon within its nucleotide context. Although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved.ResultsWe have performed an in vivo analysis of translational readthrough in mouse cells in culture using a reporter system that allows the measurement of readthrough levels as low as 10-4. We first quantified readthrough frequencies obtained with constructs carrying different codons (two Gln, two His and four Gly) immediately upstream of the stop codon. There was no effect of amino acid identity or codon frequency. However, an adenine in the -1 position was always associated with the highest readthrough levels while an uracil was always associated with the lowest readthrough levels. This could be due to an effect mediated either by the nucleotide itself or by the P-site tRNA. We then examined the importance of the downstream context using eight other constructs. No direct correlation between the +6 nucleotide and readthrough efficiency was observed.ConclusionsWe conclude that, in mouse cells, the upstream and downstream stop codon contexts affect readthrough via different mechanisms, suggesting that complex interactions take place between the mRNA and the various components of the translation termination machinery. Comparison of our results with those previously obtained in plant cells and in yeast, strongly suggests that the mechanisms involved in stop codon recognition are conserved among eukaryotes.

Sequence specificity of aminoglycoside‐induced stop codon readthrough: Potential implications for treatment of Duchenne muscular dystrophy

As a result of their ability to induce translational readthrough of stop codons, the aminoglycoside antibiotics are currently being tested for efficacy in the treatment of Duchenne muscular dystrophy patients carrying a nonsense mutation in the dystrophin gene. We have undertaken a systematic analysis of aminoglycoside-induced readthrough of each stop codon in human tissue culture cells using a dual luciferase reporter system. Significant differences in the efficiency of aminoglycoside-induced readthrough were observed, with UGA showing greater translational readthrough than UAG or UAA. Additionally, the nucleotide in the position immediately downstream from the stop codon had a significant impact on the efficiency of aminoglycoside-induced readthrough in the order C > U > A ≥ G. Our studies show that the efficiency of stop codon readthrough in the presence of aminoglycosides is inversely proportional to the efficiency of translational termination in the absence of these compounds. Using the same assay, we analyzed a 33–base pair fragment of the mouse dystrophin gene containing the mdx premature stop codon mutation UAA (A), which is also the most efficient translational terminator. The additional flanking sequences from the dystrophin gene do not significantly change the relatively low-level aminoglycoside-induced stop codon readthrough of this stop codon. The implications of these results for drug efficacy in the treatment of individual patients with Duchenne muscular dystrophy or other genetic diseases caused by nonsense mutations are discussed. Ann Neurol 2000;48:164–169

Sequence specificity of aminoglycoside-induced stop codon readthrough: Potential implications for treatment of Duchenne muscular dystrophy

As a result of their ability to induce translational readthrough of stop codons, the aminoglycoside antibiotics are currently being tested for efficacy in the treatment of Duchenne muscular dystrophy patients carrying a nonsense mutation in the dystrophin gene. We have undertaken a systematic analysis of aminoglycoside-induced readthrough of each stop codon in human tissue culture cells using a dual luciferase reporter system. Significant differences in the efficiency of aminoglycoside-induced readthrough were observed, with UGA showing greater translational readthrough than UAG or UAA. Additionally, the nucleotide in the position immediately downstream from the stop codon had a significant impact on the efficiency of aminoglycoside-induced readthrough in the order C > U > A > or = G. Our studies show that the efficiency of stop codon readthrough in the presence of aminoglycosides is inversely proportional to the efficiency of translational termination in the absence of these compounds. Using the same assay, we analyzed a 33-base pair fragment of the mouse dystrophin gene containing the mdx premature stop codon mutation UAA (A), which is also the most efficient translational terminator. The additional flanking sequences from the dystrophin gene do not significantly change the relatively low-level aminoglycoside-induced stop codon readthrough of this stop codon. The implications of these results for drug efficacy in the treatment of individual patients with Duchenne muscular dystrophy or other genetic diseases caused by nonsense mutations are discussed.

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.

Dynamics and efficiency in vivo of UGA-directed selenocysteine insertion at the ribosome.

The kinetics and efficiency of decoding of the UGA of a bacterial selenoprotein mRNA with selenocysteine has been studied in vivo. A gst-lacZ fusion, with the fdhF SECIS element ligated between the two fusion partners, gave an efficiency of read-through of 4-5%; overproduction of the selenocysteine insertion machinery increased it to 7-10%. This low efficiency is caused by termination at the UGA and not by translational barriers at the SECIS. When the selenocysteine UGA codon was replaced by UCA, and tRNASec with anticodon UGA was allowed to compete with seryl-tRNASer1 for this codon, selenocysteine was found in 7% of the protein produced. When a non-cognate SelB-tRNASec complex competed with EF-Tu for a sense codon, no effects were seen, whereas a non-cognate SelB-tRNASec competing with EF-Tu-mediated Su7-tRNA nonsense suppression of UGA interfered strongly with suppression. The induction kinetics of beta-galactosidase synthesis from fdhF'-'lacZ gene fusions in the absence or presence of SelB and/or the SECIS element, showed that there was a translational pause in the fusion containing the SECIS when SelB was present. The results show that decoding of UGA is an inefficient process and that using the third dimension of the mRNA to accommodate an additional amino acid is accompanied by considerable quantitative and kinetic costs.

Nucleotide Sequence Context Effect of a Cyclobutane Pyrimidine Dimer upon RNA Polymerase II Transcription

We have studied the role of sequence context upon RNA polymerase II arrest by a cyclobutane pyrimidine dimer using an in vitro transcription system consisting of templates containing a specifically located cyclobutane pyrimidine dimer (CPD) and purified RNA polymerase II (RNAP II) and initiation factors. We selected a model sequence containing a well characterized site for RNAP II arrest in vitro, the human histone H3.3 gene arrest site. The 13-base pair core of the arrest sequence contains two runs of T in the nontranscribed strand that impose a bend in the DNA. We hypothesized that arrest of RNAP II might be affected by the presence of a CPD, based upon the observation that a CPD located at the center of a dA6.dT6 tract eliminates bending (Wang, C.-I., and Taylor, J.-S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9072-9076). We examined the normal H3.3 sequence and a mutant sequence containing a T --> G transversion, which reduces bending and efficiency of arrest. We show that a CPD in the transcribed strand at either of two locations in the arrest site is a potent block to transcription. However, a CPD in the nontranscribed strand only transiently pauses RNAP II. The CPD in concert with a mutation in the arrest site can reduce the extent of bending of the DNA and improve readthrough efficiency. These results demonstrate the potential importance of sequence context for the effect of CPDs within transcribed sequences.

UGA suppression by tRNACmCATrp occurs in diverse virus RNAs due to a limited influence of the codon context.

We have recently identified chloroplast and cytoplasmic tRNACmCATrp as the first natural UGA suppressor tRNAs in plants. The interaction of these tRNAs with UGA involves a Cm: A mismatch at the first anticodon position. We show here that tRNACmCATrp is incapable of misreading UAA and UAG codons in vitro, implying that unconventional base pairs are not tolerated in the middle anticodon position. Furthermore, we demonstrate that the ability of tRNACmCATrp to promote UGA read-through depends on a quite simple codon context. Part of the sequence surrounding the leaky UGA stop codon in tobacco rattle virus RNA-1 was subcloned into a zein reporter gene and read-through efficiency was measured by translation of RNA transcripts in wheat germ extract. A number of mutations in the codons adjacent to the UGA were introduced by site-directed mutagenesis. It was found that single nucleotide exchanges at either side of the UGA had little effect on read-through efficiency. A pronounced influence on suppression by tRNACmCATrp was seen only if 2 or 3 nt at the 3'-side of the UGA codon had been simultaneously replaced. As a consequence of the flexible codon context accepted by tRNACmCATrp, this tRNA is able to misread the UGA in a number of plant and animal viral RNAs that use translational read-through for expression of some of their genes.

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.

Influence of the last amino acid in the nascent peptide on EF-Tu during decoding

The last two amino acids of the nascent peptide at the ribosomal P-site influence the efficiency of termination readthrough at the stop codon UGA (Mottagui-Tabar et al (1994) EMBO J 13, 249–257; Björnsson et al (1996) EMBO J 15, 1696–1704). Here we analyze this effect on readthrough by wild type or a UGA suppressor form (Su9) of tRNA Trp by varying the codons at positions −1 and −2 at the 5′ side of UGA. Strains with wild-type or mutant (ArBr) forms of elongation factor Tu (EF-Tu) were analyzed (Vijgenboom et al (1985) EMBO J 4, 1049–1052). The effect on readthrough by changing these −1 and −2 codons is different on the two forms of tRNA Trp and is also dependent on the structure of EF-Tu. Readthrough by the tRNA Trp-derived suppressor, but not wild-type tRNA Trp, is sensitive to the van der Waals volume of the last amino acid in the nascent peptide. Together with mutant EF-Tu, both forms of tRNA Trp are sensitive. The data suggest that the C-terminal amino acid in the nascent peptide is in a functional interaction with the EF-Tu ternary complex. This interaction is changed by mutation in tRNA Trp at position 24 or in EF-Tu at position 375. No indication of a changed interaction between the mutant EF-Tu and the penultimate amino acid could be found. Mutant forms of RF2 (Mikuni et al (1991) Biochimie 73, 1509–1516) and ribosomal proteins S4 and S12 (Fáxen et al (1988) J Bacteriol 170, 3756–3760) were found not to be altered in sensitivity to the last two amino acids in the nascent peptide.

Immunodetection, Expression Strategy and Complementation of Turnip Crinkle Virus p28 and p88 Replication Components

The plus-sense RNA genome of turnip crinkle virus (TCV) encodes at its 5′ end a 28-kDa protein of unspecified function. Readthrough suppression of the p28 stop codon allows for the production of an 88-kDa product which is required for genome replication. Immunological analysis of the expression of p28 and p88 demonstrated that: (i) the genome directs the synthesis of polypeptides of approximately 28 and 88 kDa, (ii) the 88-kDa protein is immunologically related to p28, consistent with p88 being a readthrough product, and (iii) p28, but not p88, is detectable in vivo. An in vivo assay, in which readthrough is linked to the expression of a β-glucuronidase reporter gene, showed that readthrough of the p28 amber stop codon occurs with an efficiency of approximately 1%. A similar efficiency of readthrough was observed when an altered context from the nonviable TCV mutant, mA2, containing a disrupted secondary structure (FfFa) spanning the p28 termination codon, was tested. This result suggests that the defective phenotype of mA2 is likely not linked to an alteration in readthrough efficiency. Additional studies demonstrated that complementation occurs in coinoculations with two nonviable TCV mutants, RT and APA, which are unable to express either p28 or p88, respectively. This result verifies that p28 is essential for TCV genome replication and provides the first definitive evidence for the role of a 5′-proximal open reading frame for any member of the family Tombusviridae.

Analysis of Translational Termination of Recombinant Human Methionyl Neurotrophin 3 in Escherichia coli

A highly efficient UGA stop codon readthrough event during the synthesis of human neurotrophin 3 in E. coli is described. The incorporation of a Trp residue at the UGA stop codon is confirmed combining both the chemical analyses and the molecular and genetic data in this report. The 3′ adjacent nuleotide to the UGA stop codon plays a crucial role in determining the readthrough efficiency in the order of A > G > C > U. The replacement of UGA with UAA or UAG totally abolished this readthrough phenomenon and the use of StpR host cells also prevented the occurrence of UGA readthrough. Gene dosage (or plasmid copy number) effect was not indicated in this event; however, the titration of RF-2 by mRNA transcripts under over-expression conditions might explain why tRNAtrp competes so well with RF-2 for UGA. Another apparently less produced readthrough product resulting from a transcript with no stop codon is also recorded, and the addition of a second in-frame stop codon increased the amount of the observed readthrough product.

The signal for translational readthrough of a UGA codon in Sindbis virus RNA involves a single cytidine residue immediately downstream of the termination codon

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.