Nonsense suppression in eukaryotes: the use of the Xenopus oocyte as an in vivo assay system

Bienz, Mariann; Kubli, Eric; Kohli, Jürg; Henau, Suzanne De; Grosjean, Henri

doi:10.1093/nar/8.22.5169

Cited by 32 publications

(14 citation statements)

References 31 publications

(1 reference statement)

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“…The aminoacylated tRNAs were further processed as described above for the Drosophila probes. Oocyte injections; extraction of the proteins Injections of X laevis oocytes and extraction of the proteins were done according to Bienz et al (1980). TYMV coat protein RNA was dissolved in distilled water and renatured for 10 min at 37°C immediately before injection.…”

Section: Methodsmentioning

confidence: 99%

“…Before addition of the sample buffer the probe was taken up in 20,l 60 mM Tris-HCl, pH 7.0, 6 mM EDTA, and neutralized with 5 M NaOH. The two histidine coat protein fragments were separated on a 20% SDS-polyacrylamide gel (Bienz et al, 1980). 10% of the coat protein remains uncleaved by CNBr and remains at the position of the intact coat protein.…”

Section: Methodsmentioning

confidence: 99%

“…Discrepancies between Xenopus oocytes and reticulocyte 'in vitro' data have been reported for suppression of the Xenopus 3-globin UAA stop codon (Bienz, 1981) and for readthrough of the rabbit 3-globin UGA stop codon by E. coli tRNATrP (Grosjean et al, 1980;un-published results). We believe that the lack of discrimination observed in the reticulocyte system is due to the failure to react to structural differences in the anticodons that are still detected by the oocyte.…”

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confidence: 99%

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Queuosine modification of the wobble base in tRNAHis influences ‘in vivo’ decoding properties.

Meier¹,

Suter²,

Grosjean³

et al. 1985

The EMBO Journal

141

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The 'in vivo' decoding properties of four tRNAHis isoacceptors, two from Drosophila melanogaster and two from brewer's yeast, were studied after their microinjection, along with turnip yellow mosaic virus (L'YMV) coat protein mRNA, into Xenopus laevis oocytes. The two Drosophila isoacceptors are identical besides containing either a guanosine (G) or the hypermodified nucleoside queuosine (Q) in the wobble position. The brewer's yeast isoacceptors differ by four bases in the anticodon stem, and by one base in the amino acceptor stem. Our results show that, under competing 'in vivo' conditions, the Drosophila tRNAHis with the anticodon GUG clearly prefers the histidine codon CAC to the codon CAU, whereas little preference is observed for the tRNAHis with the anticodon QUG for the codon CAU, and no preference for either codon by the two yeast isoacceptors. Hence, it can be concluded that the presence of the Q-base clearly affects the choice of the codon. This is the first demonstration of an 'in vivo' codon preference by tRNA isoacceptors differing in the modification of the wobble base during the elongation step of protein synthesis. These results imply that one function of the Q-base is at the translational level. Key words: decoding/modification/queuosine/transfer RNA/ wobble base as a sensitive assay system. The two Drosophila tRNAHis differ only by the presence or absence of the Q-base in the wobble position (Altwegg and and unpublished results), whereas the two brewer's yeast tRNAs differ by four bases in the anticodon stem and one base in the amino acid acceptor stem. They do not contain the Q-modification in the wobble position, i.e., they possess identical anticodons (Keith et al., 1983).Turnip yellow mosaic virus (TYMV) coat protein RNA contains three histidine codons (Figure 1): two CAU in the 5' and one CAC in the 3' half of the mRNA (Guilley and Briand, 1978). Cyanogen bromide cleavage of the coat protein at the methionine residues yields three peptides: one large fragment (X) of 135 amino acid residues, containing the two CAU coded histidines, one fragment of 42 amino acids containing no histidine, and a small fragment (Y) of 10 amino acid residues, containing the unique CAC coded histidine. Thus, it is possible to study the incorporation of histidine from each of the two tRNAH1s isoacceptors into the coat protein sites coded by the two histidine codons under competing conditions. TYMV coat protein mRNA, histidinol (inhibits the endogenous synthetase, Hansen et al., 1972), and equal amounts (pmol) of aminoacylated tRNA isoacceptors were injected into Xenopus oocytes and, after 4 h of incubation, the incorporation into the peptides X and Y determined.Since only [3H]histidine labelling provides the necessary specific activity needed for these experiments, only one of the isoacceptors could be labeled. The competing isoacceptor carried an unlabeled histidine. In order to exclude potential artefacts (e.g., heterogeneity of the oocytes) we calculated the percentages and ratios of histidine incorporation f...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Queuosine modification of the wobble base in tRNAHis influences ‘in vivo’ decoding properties.

Meier¹,

Suter²,

Grosjean³

et al. 1985

The EMBO Journal

141

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“…6 ) UGA suppressor activity. As described previously [19], samples of five X . laevis oocytes were injected with 5 ng rabbit globin mRNA, prepared according to Marbaix et al [20], and variable concentrations of tRNACys UCA as shown in Fig.…”

Section: Construction Of Trnacys (Anticodon Uca)mentioning

confidence: 99%

“…The Xenopus oocyte has previously been shown to be a sensitive system in which to analyse nonsense suppression activity in vivo [19,241. UGA suppressor activity may be assayed by the co-injection of the messenger for rabbit pglobin, which is terminated by a UGA codon, and the putative suppressor tRNA.…”

Section: U G a Suppressor Activity Of Trnacys (Anticodon U C A )mentioning

confidence: 99%

Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNA^Cys

Vacher

Grosjean

Henau

et al. 1984

European Journal of Biochemistry

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In this paper we describe the construction of a yeast tRNACys UGA suppressor. After specific hydrolysis of the parent molecule, the first base of the anticodon GCA was replaced by a uracil. The resulting molecule, harboring a UCA anticodon, was injected into Xenopus laevis oocytes in order to test its biological activities. The level of aminoacylation was similar to that of the parent molecule. Readthrough of the UGA termination codon in /?-globin mRNA, coinjected with the tRNA, indicated suppressor activity; however, tRNACys (anticodon UCA) was a much less efficient suppressor than others tested under the same conditions. We see no post-transcriptional modification of the uracil in the anticodon wobble position after injection into oocytes. This may be related to the low suppressor activity; however, it is also possible that other features of tRNACyS structure may be unadapted to efficient UCA anticodon function.Several lines of evidence now suggest that the overall functional efficiency of a tRNA molecule in protein synthesis is dependent on the structure of the whole of the molecule and its relation to the anticodon. Thus Gorini, observing the varying efficiencies of different nonsense suppressors remarked [I] "it appears that a 'correct' anticodon obtained by mutation can be out of context in the resulting tRNA suppressor molecule". In addition to the occurrence of invariant nucleotides in the tRNA molecule, it is known that base usage in other positions is often far from random [2]. One of the most versatile ways of investigating the importance of particular bases or regions of the molecule is to construct tRNAs with modified primary structure. This may be done either by modification of the DNA or the RNA sequence. Temple et al. have recently reported the construction of an active suppressor tRNALy" by site-directed mutagenesis of the gene [3]. By modification of the RNA sequence, Bruce et al. have succeeded in synthesising a biologically active amber suppressor by modification at the RNA level of yeast tRNAPhe [4]. Here we describe the application of the RNA mutagenic technology to the construction of a potential UGA suppressor with anticodon UCA, by base substitution in yeast tRNACyS. This methodology, dependent largely on the properties of RNA ligase and polynucleotide kinase from phage T4 [5, 61, was initially developed by the group of Uhlenbeck, and applied successfully to the modification of several yeast tRNAs: tRNAPhe [4], tRNAASp [8] and tRNAkg [9].Essentially three reasons led us to select yeast tRNACy" as an interesting species for modification. Among the anticodon changes that may be easily introduced, one should lead to a UGA suppressor species, the activity of which may be readily assayed in vivo as well as in vitro, The normal tRNACYs anticodon, GCA, is conveniently split by ribonuclease TI. Finally, the availability of high specific activity [35S]cysteine should facilitate characterisation of polypeptides synthesized in vivo or in vitro.Enzymes. RNAse TI (EC 3.1.27.3); nuclease S1 (EC 3.1.30...

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Therapeutic promise of engineered nonsense suppressor tRNAs

2021

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Nonsense mutations change an amino acid codon to a premature termination codon (PTC) generally through a single‐nucleotide substitution. The generation of a PTC results in a defective truncated protein and often in severe forms of disease. Because of the exceedingly high prevalence of nonsense‐associated diseases and a unifying mechanism, there has been a concerted effort to identify PTC therapeutics. Most clinical trials for PTC therapeutics have been conducted with small molecules that promote PTC read through and incorporation of a near‐cognate amino acid. However, there is a need for PTC suppression agents that recode PTCs with the correct amino acid while being applicable to PTC mutations in many different genomic landscapes. With these characteristics, a single therapeutic will be able to treat several disease‐causing PTCs. In this review, we will focus on the use of nonsense suppression technologies, in particular, suppressor tRNAs (sup‐tRNAs), as possible therapeutics for correcting PTCs. Sup‐tRNAs have many attractive qualities as possible therapeutic agents although there are knowledge gaps on their function in mammalian cells and technical hurdles that need to be overcome before their promise is realized. This article is categorized under: RNA Processing > tRNA Processing Translation > Translation Regulation

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Nonsense suppression in eukaryotes: the use of the Xenopus oocyte as an in vivo assay system

Cited by 32 publications

References 31 publications

Queuosine modification of the wobble base in tRNAHis influences ‘in vivo’ decoding properties.

Queuosine modification of the wobble base in tRNAHis influences ‘in vivo’ decoding properties.

Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNA^Cys

Therapeutic promise of engineered nonsense suppressor tRNAs

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Nonsense suppression in eukaryotes: the use of the Xenopus oocyte as an in vivo assay system

Cited by 32 publications

References 31 publications

Queuosine modification of the wobble base in tRNAHis influences ‘in vivo’ decoding properties.

Queuosine modification of the wobble base in tRNAHis influences ‘in vivo’ decoding properties.

Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNACys

Therapeutic promise of engineered nonsense suppressor tRNAs

Contact Info

Product

Resources

About

Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNA^Cys