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At least 19 recordsLinked to original sources
PubMed1998

The association of nonsense codons with exon skipping.

Some genes that contain premature nonsense codons express alternatively-spliced mRNA that has skipped the exon containing the nonsense codon. This paradoxical association of translation signals (nonsense codons) and RNA splicing has inspired numerous explanations. The first is based on the fact that premature nonsense codons often reduce mRNA abundance. The reduction in abundance of full-length mRNA then allows more efficient amplification during PCR of normal, minor, exon-deleted products. This mechanism has been demonstrated to explain an extensive correlation between nonsense codons and exon-skipping for the hamster Hprt gene. The second explanation is that the mutation producing an in-frame nonsense codon has an effect on exon definition. This has been demonstrated for the Mup and hamster Hprt gene by virtue of the fact that missense mutations at the same sites also are associated with the same exon-deleted mRNA. The third general explanation is that a hypothetical process takes place in the nucleus that recognizes nonsense codons, termed 'nuclear scanning', which then has an effect on mRNA splicing. Definitive evidence for nuclear scanning is lacking. My analysis of both nonsense and missense mutations associated with exon skipping in a large number of genes revealed that both types of mutations frequently introduce a T into a purine-rich DNA sequence and are often within 30 base pairs of the nearest exon boundary. This is intriguing given that purine-rich splicing enhancers are known to be inhibited by the introduction of a T. Almost all mutations associated with exon skipping occur in purine-rich or A/C-rich sequences, also characteristics of splicing enhancers. I conclude that most cases of exon skipping associated with premature termination codons may be adequately explained either by a structural effect on exon definition or by nonquantitative methods to measure mRNA, rather than an effect on a putative nuclear scanning mechanism.

Animals↗
PubMed1996

Evidence that the decay of nucleus-associated nonsense mRNA for human triosephosphate isomerase involves nonsense codon recognition after splicing.

For most of the mammalian mRNAs that have been shown to be reduced in abundance by a nonsense or a frameshift mutation that generates a nonsense codon, reduction takes place while the mRNA is nucleus-associated rather than after the mRNA has been exported to the cytoplasm (reviewed in Maquat LE, 1995, RNA 1:453-465). A variety of mechanisms have been put forth to explain how a nonsense codon could affect the abundance of nuclear mRNA. Some mechanisms have implicated nonsense codon recognition in the nucleus prior to splicing. Among the best-studied nonsense transcripts that manifest nonsense-mediated alterations in nucleus-associated metabolism are those that derive from human alleles for the glycolytic enzyme triosephosphate isomerase (TPI). Nonsense codons within TPI transcripts have been shown to reduce the half-life of completely spliced TPI (mRNA that co-purifies with nuclei (Belgrader P et al., 1994, Mol Cell Biol 14:8219-8228). However, whether or not nonsense codon recognition within TPI transcripts takes place prior to or after splicing remained unresolved. To address this issue, codons that span two exons, i.e., are disrupted by an intron prior to pre-mRNA splicing, were converted to nonsense. If nonsense codon recognition were to precede splicing, then the disrupting intron would be expected to preclude nonsense codon recognition by preventing the physical juxtapositioning of the codon nucleotides. In the absence of nonsense codon recognition, there would be no nonsense-mediated reduction in TPI mRNA abundance. The results of northern (RNA) blot hybridization demonstrated that the two nonsense codons of this type that were studied reduced the level of total, nuclear and cytoplasmic TPI mRNA to an average of 12% of normal, consistent with each nonsense codon being competent to mediate nuclear mRNA degradation. The possibility that the nonsense codons reduced TPI mRNA abundance by altering TPI mRNA abundance or splicing was eliminated by using RT-PCR to demonstrate that the level of each intron within pre-mRNA was essentially unaffected and cDNA sequencing to demonstrate that splice site choice was unaltered. Furthermore, missense codons that harbored some of the nonsense codon changes were found to have little effect on mRNA abundance. These findings, plus the previous finding that a suppressor tRNA abrogates the decay of TPI mRNA brought about by a nonsense codon residing within a single exon (Belgrader P, Cheng J, Maquat LE, 1993, Proc Natl Acad Sci USA 90:482-486), argue strongly that nonsense codon recognition in the nonsense-mediated decay of TPI mRNA takes place after splicing.

Base Sequence↗
PubMed1996

Premature nonsense codons decrease the stability of phytohemagglutinin mRNA in a position-dependent manner.

Premature termination of translation has often been associated with decreased mRNA accumulation in plants, but the affected step in gene expression has not been identified. To investigate this problem, the expression of wild-type and mutant alleles of the bean phytohemagglutinin (PHA) gene has been examined in tobacco cells and transgenic plants. Measurement of mRNA decay rates in stably transformed cell lines demonstrated that premature nonsense codons markedly destabilized the mRNA. This decreased stability was also reflected by decreased accumulation of transcripts containing premature nonsense codons in transgenic plants. The positional dependence of the nonsense codon effect was evaluated by introducing premature nonsense codons at different distances from the PHA AUG start codon. Transcripts with nonsense codons about 20, 40 or 60% of the way through the normal PHA coding region yielded highly unstable mRNAs, whereas a transcript with a nonsense codon at 80% was as stable as wild-type. The ability to recognize and rapidly degrade certain transcripts with early nonsense codons could provide plant cells with a means to minimize the production of wasteful and possible deleterious truncated proteins.

Alleles↗
PubMed1999

Infrequent translation of a nonsense codon is sufficient to decrease mRNA level.

In many organisms nonsense mutations decrease the level of mRNA. In the case of mammalian cells, it is still controversial whether translation is required for this nonsense-mediated RNA decrease (NMD). Although previous analyzes have shown that conditions that impede translation termination at nonsense codons also prevent NMD, the residual level of termination was unknown in these experiments. Moreover, the conditions used to impede termination might also have interfered with NMD in other ways. Because of these uncertainties, we have tested the effects of limiting translation of a nonsense codon in a different way, using two mutations in the immunoglobulin mu heavy chain gene. For this purpose we exploited an exceptional nonsense mutation at codon 3, which efficiently terminates translation but nonetheless maintains a high level of mu mRNA. We have shown 1) that translation of Ter462 in the double mutant occurs at only approximately 4% the normal frequency, and 2) that Ter462 in cis with Ter3 can induce NMD. That is, translation of Ter462 at this low (4%) frequency is sufficient to induce NMD.

Animals↗
PubMed2003

The effect of nonsense codons on splicing: a genomic analysis.

The phenomenon of nonsense-associated altered splicing raises the possibility that the recognition of in-frame nonsense codons is used generally for exon identification during pre-mRNA splicing. However, nonsense codon frequencies in pseudo exons and in regions flanking 5' splice sites are no greater than that expected by chance, arguing against the widespread use of this strategy as a means of rejecting potential splice sites.

Codon, Nonsense↗
PubMed1978

Context effects on nonsense codon suppression in Escherichia coli.

The influence of mRNA context on nonsense codon suppression has been studied by suppression measurements at one site in the Escherichia coli trpE gene and at two sites in the trpA gene. The ratio of suppression efficiencies of amber and ochre codons at each site (homotopic pairs) has been compared using ochre suppressing derivatives of tRNATyr. This ratio is independent of differential effects of the inserted amino acid on enzyme function. We have found that mRNA context can change the ratio of suppression efficiencies of homotopic nonsense codons at the three sites in the trp gene system over a ten-fold range. The causes of such variation, and, in particular the effect of certain adjacent nucleotides on nonsense codon suppression are considered.

Anthranilate Synthase↗
PubMed1994

Mammalian nonsense codons can be cis effectors of nuclear mRNA half-life.

Frameshift and nonsense mutations within the gene for human triosephosphate isomerase (TPI) that generate a nonsense codon within the first three-fourths of the protein coding region have been found to reduce the abundance of the product mRNA that copurifies with nuclei. The cellular process and location of the nonsense codon-mediated reduction have proven difficult to elucidate for technical reasons. We show here, using electron microscopy to judge the purity of isolated nuclei, that the previously established reduction to 25% of the normal mRNA level is evident for nuclei that are free of detectable cytoplasmic contamination. Therefore, the reduction is likely to be characteristic of bona fide nuclear RNA. Fully spliced nuclear mRNA is identified by Northern (RNA) blot hybridization and a reverse transcription-PCR assay as the species that undergoes decay in experiments that used the human c-fos promoter to elicit a burst and subsequent shutoff of TPI gene transcription upon the addition of serum to serum-deprived cells. Finally, the finding that deletion of a 5' splice site of the TPI gene results predominantly but not exclusively in the removal by splicing (i.e., skipping) of the upstream exon as a part of the flanking introns has been used to demonstrate that decay is specific to those mRNA products that maintain the nonsense codon. This result, together with our previous results that implicate translation by ribosomes and charged tRNAs in the decay mechanism, indicate that nonsense codon recognition takes place after splicing and triggers decay solely in cis. The possibility that decay takes place during the process of mRNA export from the nucleus to the cytoplasm is discussed.

Animals↗
PubMed1983

The role of 2-methylthio-N6-isopentenyladenosine in readthrough and suppression of nonsense codons in Escherichia coli.

Readthrough and suppression of nonsense codons was compared in Escherichia coli strains with and without a miaA mutation, which confers a loss of the isopentenyladenosine modification in transfer RNA. Generally speaking, our results conform to predictions based on previous literature. In addition, we showed that the miaA mutation in strain TRPX is itself a UAA mutation. An antagonism between miaA and rpsL mutations, which confer streptomycin resistance, was also discovered. Our data further suggest that slight alterations of the translation apparatus are easily detectable by monitoring readthrough and suppression of nonsense codons. Our findings are discussed in the context of old and recent reports.

Adenosine↗
PubMed1993

Evidence to implicate translation by ribosomes in the mechanism by which nonsense codons reduce the nuclear level of human triosephosphate isomerase mRNA.

The abundance of the mRNA for human triosephosphate isomerase (TPI) is decreased to 20-30% of normal by frameshift and nonsense mutations that prematurely terminate translation within the first three-quarters of the reading frame. The decrease has been shown to be attributable to a reduced level of TPI mRNA that copurifies with nuclei. Given that the translational reading frame of an mRNA is assessed in the cytoplasm during protein synthesis, cytoplasmic and nuclear RNA processes may be linked. Alternatively, a nuclear mechanism may exist whereby in-frame nonsense codons can be identified. To differentiate between these two possibilities, two distinct modulators of protein synthesis have been tested for the ability to influence the nonsense-codon-mediated reduction in the mRNA level. (i) A suppressor tRNA, which acts in trans to suppress an amber nonsense codon within TPI mRNA, and (ii) a hairpin structure in the 5' untranslated region of TPI mRNA, which acts exclusively in cis to inhibit initiation of TPI mRNA translation, were found, individually, and to a greater extent, together, to abrogate the decrease in mRNA. These results show that tRNA and ribosomes coordinately mediate the effect of a nonsense codon on the level of newly synthesized TPI mRNA. We suggest that the premature termination of TPI mRNA translation in the cytoplasm can reduce the level of TPI mRNA that fractionates with nuclei.

Animals↗
PubMed2003

Glutamine is incorporated at the nonsense codons UAG and UAA in a suppressor-free Escherichia coli strain.

Readthrough of the nonsense codons UAG, UAA, and UGA is seen in Escherichia coli strains lacking tRNA suppressors. Earlier results indicate that UGA is miscoded by tRNA(Trp). It has also been shown that tRNA(Tyr) and tRNA(Gln) are involved in UAG and UAA decoding in several eukaryotic viruses as well as in yeast. Here we have investigated which amino acid(s) is inserted in response to the nonsense codons UAG and UAA in E. coli. To do this, the stop codon in question was introduced into the staphylococcal protein A gene. Protein A binds to IgG, which facilitates purification of the readthrough product. We have shown that the stop codons UAG and UAA direct insertion of glutamine, indicating that tRNA(Gln) can read the two codons. We have also confirmed that tryptophan is inserted in response to UGA, suggesting that it is read by tRNA(Trp).

Codon, Nonsense↗
PubMed1995

When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells.

It appears that no organism is immune to the effects of nonsense codons on mRNA abundance. The study of how nonsense codons alter RNA metabolism is still at an early stage, and our current understanding derives more from incidental vignettes than from experimental undertakings that address molecular mechanisms. Challenges for the future include identifying the gene products and RNA sequences that function in nonsense mediated RNA loss, resolving the cause and consequences of there apparently being more than one cellular site and mechanism for nonsense-mediated RNA loss, and understanding how these sites and mechanisms are related to both constitutive and specialized pathways of pre-mRNA processing and mRNA decay.

Animals↗
PubMed1994

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.

Codon↗
PubMed1994

Nonsense codon mutations in the terminal exon of the beta-globin gene are not associated with a reduction in beta-mRNA accumulation: a mechanism for the phenotype of dominant beta-thalassemia.

We present in vivo evidence that there is no reduction in beta-mRNA accumulation in patients with nonsense codons in the terminal exon of the beta-globin gene. Using reverse transcriptase/polymerase chain reaction (RT-PCR), beta-globin cDNA was isolated from the reticulocytes of individuals heterozygous for nonsense codon mutations in exons II and III of the beta-globin gene. Clinically asymptomatic individuals heterozygous for mutations causing premature termination of translation in exon II [beta(0)39(C-T) and F/S71/72(+A)] were found to have almost no mutant beta-cDNA, whereas patients with nonsense codon mutations in exon III [beta 121(G-T) and beta 127(C-T)] with the clinical phenotype of thalassemia intermedia had comparable levels of mutant and normal beta-cDNA. Translation of the mutant beta-mRNA from patients with nonsense codon mutations in exon III would give rise to truncated beta-globin chains, which could explain the more severe phenotype seen in these individuals.

Adolescent↗
PubMed1992

Nonsense-codon mutations of the ornithine aminotransferase gene with decreased levels of mutant mRNA in gyrate atrophy.

A generalized deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT) is the inborn error in gyrate atrophy (GA), an autosomal recessive degenerative disease of the retina and choroid of the eye. Mutations in the OAT gene show a high degree of molecular heterogeneity in GA, reflecting the genetic heterogeneity in this disease. Using the combined techniques of PCR, denaturing gradient gel electrophoresis, and direct sequencing, we have identified three nonsense-codon mutations and one nonsense codon-generating mutation of the OAT gene in GA pedigrees. Three of them are single-base substitutions, and one is a 2-bp deletion resulting in a reading frameshift. A nonsense codon created at position 79 (TGA) by a frameshift and nonsense mutations at codons 209 (TAT----TAA) and 299 (TAC----TAG) result in abnormally low levels of OAT mRNA in the patient's skin fibroblasts. A nonsense mutation at codon 426 (CGA----TGA) in the last exon, however, has little effect on the mRNA level. Thus, the mRNA level can be reduced by nonsense-codon mutations, but the position of the mutation may be important, with earlier premature-translation termination having a greater effect than a later mutation.

Adult↗
PubMed1998

A splice-site mutation that induces exon skipping and reduction in lysyl hydroxylase mRNA levels but does not create a nonsense codon in Ehlers-Danlos syndrome type VI.

The type VI variant of Ehlers-Danlos syndrome (EDS) is a heritable connective tissue disorder caused by a deficiency in the activity of lysyl hydroxylase, an enzyme required for the post-translational processing of collagens. We have characterized a novel type of mutation in a young female patient with type VI EDS, in which cells possess only 12% of the lysyl hydroxylase activity that is detected in unaffected cells. The syndrome was found to be caused by a homozygous insertion of two thymidines at the 5' splice site consensus sequence of intron 9 in the lysyl hydroxylase gene. The insertion interfered with normal splicing of the primary RNA transcript and resulted in an inframe deletion of the 132 nucleotides coded by exon 9 from the lysyl hydroxylase mRNA. In addition, the mutation caused a marked reduction in the steady-state level of the truncated mRNA, which was less than 15% of the level found in unaffected cells. The mutation also reduced the amount of the enzyme protein produced, which was estimated to be about 20% of that in control cells. However, the mutation did not affect the stability of the abnormally spliced mRNA nor the normal localization of the enzyme protein in the endoplasmic reticulum. According to our results, the reduction in enzymatic activity observed in this patient is caused by low levels of both lysyl hydroxylase mRNA and enzyme protein. The primary cellular defect associated with this mutation, therefore, appears to be at the level of nuclear mRNA metabolism even though the mutation did not create a premature translation termination codon.

Codon, Nonsense↗
PubMed1999

Facile characterization of translation initiation via nonsense codon suppression.

A new strategy for studying the mechanism of translation initiation in eukaryotes has been developed. The strategy involves the use of an in vitro translation system to incorporate a non-natural fluorescent amino acid into a protein from a suppressor tRNAPheCUA misacylated with that amino acid. It is thereby possible to monitor translation initiation efficiency at an AUG codon in different contexts; this is illustrated for three constructs encoding Escherichia coli dihydrofolate reductase mRNA with different translation initiation regions. Fluorescence measurements after in vitro translation of the mRNAs in rabbit reticulocyte lysate reflected differences in the position and efficiency of translation initiation and, therefore, can be used for characterization of the translation initiation process.

Amino Acid Sequence↗
PubMed2003

Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli.

The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA(Ser)(CGA), an Arabidopsis intron-containing tRNA(Tyr)(GTA) and an Arabidopsis intron-containing tRNA(Met)(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of beta-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA(Ser) gene and the GUS reporter gene resulted in approximately 10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA(Tyr) or tRNA(Met) genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA(Met)(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA(Tyr) to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA(Tyr) and tRNA(Met) were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.

Arabidopsis↗
PubMed1996

Nondefective rotavirus mutants with an NSP1 gene which has a deletion of 500 nucleotides, including a cysteine-rich zinc finger motif-encoding region (nucleotides 156 to 248), or which has a nonsense codon at nucleotides 153-155.

We isolated two nondefective bovine rotavirus mutants (A5-10 and A5-16 clones) which have nonsense mutations in the early portion of the open reading frame of the NSP1 gene. In the NSP1 gene (1,587 bases long) of A5-10, a nonsense codon is present at nucleotides 153 to 155 just upstream of the coding region (nucleotides 156 to 230) of a cysteine-rich Zn finger motif. A5-16 gene 5 (1,087 bases long) was found to have a large deletion of 500 bases corresponding to nucleotides 142 to 641 of a parent A5-10 NSP1 gene and to have a nonsense codon at nucleotides 183 to 185, which resulted from the deletion. Expression of gene 5-specific NSP1 could not be detected in MA-104 cells infected with the A5-10 or A5-16 clone or in an in vitro translation system using the plasmids with gene 5 cDNA from A5-10 or A5-16. Nevertheless, both A5-10 and A5-16 replicated well in cultured cells, although the plaque size of A5-16 was extremely small.

Amino Acid Sequence↗