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

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

New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae

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 tRNA^Gln 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.     

Construction and expression of nonsense suppressor tRNAs which function in plant cells

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.     

When nonsense makes sense and vice versa: Noncanonical decoding events at stop codons in eukaryotes

Regulation of protein synthesis at the level of translation termination is a relatively underexplored, but rapidly expanding field. Recent advances in elucidating the mechanism of translation termination are helping to understand noncanonical events associated with translation termination. These “recording” events include read-through of stop codons, insertion of unusual amino acids such as selenocysteine, and production of several polypeptides from one open reading frame. This review summarizes data on termination-dependent recording events and proposes that there are two types of stop codon-associated sequences optimized to perform different functions: termination of translation per se or alternative elongation events. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 731–741. The article is published in the original.     

Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.

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.     

Dramatic events in ciliate evolution: alteration of UAA and UAG termination codons to glutamine codons due to anticodon mutations in two Tetrahymena tRNAs

The three major glutamine tRNAs of Tetrahymena thermophila were isolated and their nucleotide sequences determined by post-labeling techniques. Two of these tRNAs^Gln show unusual codon recognition: a previously isolated tRNA^Gln_UmUA and a second species with CUA in the anticodon (tRNA^Gln_CUA). These two tRNAs recognize two of the three termination codons on natural mRNAs in a reticulocyte system. tRNA^Gln_UmUA reads the UAA codon of α-globin mRNA and the UAG codon of tobacco mosaic virus (TMV) RNA, whereas tRNA^Gln_CUA recognizes only UAG. This indicates that Tetrahymena uses UAA and UAG as glutamine codons and that UGA may be the only functional termination codon. A notable feature of these two tRNAs^Gln is their unusually strong readthrough efficiency, e.g. purified tRNA^Gln_CUA achieves complete readthrough over the UAG stop codon of TMV RNA. The third major tRNA^Gln of Tetrahymena has a UmUG anticodon and presumably reads the two normal glutamine codons CAA and CAG. The sequence homology between tRNA^Gln_UmUG and tRNA^Gln_UmUA is 81%, whereas that between tRNA^Gln_CUA and tRNA^Gln_UmUA is 95%, indicating that the two unusual tRNAs^Gln evolved from the normal tRNA^Gln early in ciliate evolution. Possible events leading to an altered genetic code in ciliates are discussed.     

Non-canonical decoding events at stop codons in eukaryotes

Regulation of protein synthesis at translation termination is a relatively under-explored, but rapidly expanding field. Recent advances in elucidating the mechanism of translation termination are helping to understand non-canonical events associated with translation termination. These "recoding" events include read-through of stop-codons, insertion of unusual amino acids such as selenocysteine and production of several polypeptides from one open reading frame. This review summarises data on termination-dependent recoding events, and proposes that there are two types of stop codon-associated sequences optimized to perform different functions: termination of translation per se or alternative elongation events.     

Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs

Incorporation of unnatural amino acids into proteins in vivo, known as expanding the genetic code, is a useful technology in the pharmaceutical and biotechnology industries. This procedure requires an orthogonal suppressor tRNA that is uniquely acylated with the desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. In order to enhance the numbers and types of suppressor tRNAs available for engineering genetic codes, we have developed a convenient screening system to generate suppressor tRNAs with good orthogonality from the available library of suppressor tRNA mutants. While developing an amber suppressor tRNA, we discovered that amber suppressor tRNA with poor orthogonality inhibited the growth rate of the host, indicating that suppressor tRNA demonstrates a species-specific toxicity to host cells. We verified this species-specific toxicity using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaea. We also confirmed that adding terminal CCA to Methanococcus jannaschii tRNA^Tyr mutant is important to its toxicity against Escherichia coli. Further, we compared the toxicity of the suppressor tRNA toward the host with differing copy numbers. Using the combined toxicity of suppressor tRNA toward the host with blue–white selection, we developed a convenient screening system for orthogonal suppressor tRNA that could serve as a general platform for generating tRNA/aaRS pairs and thereby obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNA^Tyr.     

Reversion of trpA nonsense mutations by deletion of the chain-termination codons

This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of Escherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of selecting for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined. trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a frequency of approximately 2 per 10(9) viable cells. They were identical in that they both resulted from a 3 base-pair deletion, namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model multienzyme complex, and for the study of mechanisms by which deletions can be generated.     

Binary specification of nonsense codons by splicing and cytoplasmic translation.

Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.     

Suppressors of lysine codons may be misacylated lysine tRNAs

We describe a novel class of missense suppressors that read the codons for lysine at two positions (211 and 234) in the trpA polypeptide of Escherichia coli. The suppressor mutations are highly linked to lysT, a gene for lysine tRNA. The results suggest that the suppressors are misacylated lysine tRNAs that carry glycine or alanine. The mutant codons are apparently suppressed better at position 211 than at position 234, indicating the existence of codon context effects in missense suppression.     

Altered discrimination of start codons and initiator tRNAs by mutant initiation factor 3.

IF3 is essential for ensuring the fidelity of the initiation step of translation in bacterial cells. Mutations at residues R99 and R131 in the C-terminal domain of the factor have previously been shown to increase initiation from the non-canonical GUA codon. Here we show that these mutant forms of IF3 fail to discriminate against initiation from many different non-AUG codons. They also enhance the activity of mutant tRNAs carrying changes in the three consecutive G-C pairs that are conserved in the anticodon stem of initiator tRNAs. In addition, the IF3 mutants stimulate initiations from leaderless mRNAs and from internal initiation codons, in the absence of any SD-anti-SD interaction. These results indicate that IF3 ensures the accuracy of initiation by inspecting both the codon-anticodon pairing and unique features of the initiator tRNA as well as suppressing initiation from other potential start sites within the mRNA.     

Effects of surrounding sequence on the suppression of nonsense codons

Using a lacI-Z fusion system, we have determined the efficiency of suppression of nonsense codons in the I gene of Escherichia coli by assaying beta-galactosidase activity. We examined the efficiency of four amber suppressors acting on 42 different amber (UAG) codons at known positions in the I gene, and the efficiency of a UAG suppressor at 14 different UGA codons. The largest effects were found with the amber suppressor supE (Su2), which displayed efficiencies that varied over a 35-fold range, and with the UGA suppressor, which displayed a 170-fold variation in efficiency. Certain UGA sites were so poorly suppressed (less than 0.2%) by the UGA suppressor that they were not originally detected as nonsense mutations. Suppression efficiency can be correlated with the sequence on the 3' side of the codon being suppressed, and in many cases with the first base on the 3' side. In general, codons followed by A or G are well suppressed, and codons followed by U or C are poorly suppressed. There are exceptions, however, since codons followed by CUG or CUC are well suppressed. Models explaining the effect of the surrounding sequence on suppression efficiency are considered in the Discussion and in the accompanying paper.