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PubMed1991

Nonsense codons within the Rous sarcoma virus gag gene decrease the stability of unspliced viral RNA.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. In contrast, the levels of spliced viral RNAs were not affected in our transient expression assays in chicken cells. Experiments using the transcription inhibitor dactinomycin showed that mutant unspliced RNAs were degraded more rapidly than wild-type RNA. Furthermore, mutant RNAs could be partially stabilized by coexpression of wild-type gag proteins in trans; however, intact gag proteins were not required to maintain the stability of RNAs which did not contain premature termination codons. Thus, termination codons seemed to destabilize the RNA not because of their effect on gag protein function but instead because they disrupted the process of translating the gag region of the RNA. Analysis of double-mutant constructs containing both deletions and termination codons within the gag gene also suggested that the stability of the unspliced RNA was affected by a cis-acting interaction between the RNA and ribosomes.

Animals↗

PubMed1975

Synthesis of a chemically reactive analog of the nonsense codon U-G-A. Its reaction with ribosomes of Escherichia coli.

Nitrophenylated 5'-uridylic acid could be employed as primer in a polyribonucleotide nucleotidyltransferase (Micrococcus luteus) reaction to yield 5'-nitrophenylated Pu-G-A. After reduction of the nitrophenyl moiety and subsequent bromoacetylation, a 5'-bromoacetamido-phenyl-phosphorylated U-G-A was obtained, which could be used as an affinity label for the ribosomal binding site of the nonsense codon. If freshly prepared active ribosomes were employed in the incubation mixtures, the U-G-A analog reacted exclusively with one protein, which is tentatively identified as protein S18. Exposure of ribosomes to low temperatures gave rise to a reaction of the U-G-A label with another protein, which was identified as protein S4, the ram gene product. The results of the affinity labeling experiments with the chemically reactive U-G-A derivative are very similar to that obtained with a corresponding derivative of the initiation codon A-U-G (O. Pongs and E. Lanka (1975) Proc. Natl. Acad. Sci. U.S.A. 75, 1505-1509), which suggests that 70S ribosomes have one preferential codon binding site.

Codon↗

PubMed2001

Precursor RNAs harboring nonsense codons accumulate near the site of transcription.

Messenger RNAs containing premature termination codons (PTCs) are selectively eliminated by nonsense-mediated mRNA decay (NMD). Paradoxically, although cytoplasmic ribosomes are the only known species capable of PTC recognition, in mammals many PTC-containing mRNAs are apparently eliminated prior to release from the nucleus. To determine whether PTCs can influence events within the nucleus proper, we studied the immunoglobulin (Ig)-mu and T cell receptor (TCR)-beta genes using fluorescent in situ hybridization (FISH). Alleles containing PTCs, but not those containing a missense mutation or a frameshift followed by frame-correcting mutations, exhibited elevated levels of pre-mRNA, which accumulated at or near the site of transcription. Our data indicate that mRNA reading frame can influence events at or near the site of gene transcription.

Animals↗

PubMed1980

Yeast mitochondrial tRNATrp can recognize the nonsense codon UGA.

DNA sequence analysis of mitochondrial genes that code for some mitochondrial proteins has suggested that the opal terminator, UGA, is used as a sense codon in mitochondria. The complete sequences of both the yeast and human genes coding for cytochrome oxidase subunit II contain UGA codons in the reading frame. When the protein sequences predicted by these DNA sequences are compared with the known protein sequence of bovine mitochondrial cytochrome oxidase subunit II, there are regions of homology, in which UGA codons correspond to tryptophan residues. Therefore it has been suggested that UGA specifies tryptophan in the mitochondrial code. We have isolated a yeast mitochondrial tRNATrp and used it to locate the mitochondrial tRNATrp gene in pBR322-mitochondrial DNA recombinants. DNA sequence analysis of this gene revealed that the mitochondrial tRNATrp anticodon is 5'UCA3'. Because there is a U in the wobble position, this tRNA can recognize and insert tryptophan into a growing polypeptide chain in response to the nonsense codon UGA.

Anticodon↗

PubMed1997

The Frequency and Degree of Cosuppression by Sense Chalcone Synthase Transgenes Are Dependent on Transgene Promoter Strength and Are Reduced by Premature Nonsense Codons in the Transgene Coding Sequence.

By comparing the effects of strong and weak promoters that drive sense chalcone synthase (Chs) transgenes in large populations of independently transformed plants, we show here that a strong transgene promoter is required for high-frequency cosuppression of Chs genes and for production of the full range of cosuppression phenotypes. In addition, sense Chs transgenes driven by a cauliflower mosaic virus 35S promoter possessing a single copy of the upstream activator region (UAR) were found to produce a significantly lower degree of cosuppression than they did when the transgene promoter possessed two or four copies of the UAR. It has been shown elsewhere that 35S promoter strength increases with increasing UAR copy number. Frameshift mutations producing early nonsense codons in the Chs transgene were found to reduce the frequency and the degree of cosuppression. These results suggest that promoter strength and transcript stability determine the degree of cosuppression, supporting the hypothesis that sense cosuppression is a response to the accumulation of transcripts at high concentrations. This conclusion was shown to apply to single-copy transgenes but not necessarily to inversely repeated transgenes. The results presented here also have significance for efficient engineering of cosuppression phenotypes for use in research and agriculture.

Journal Article↗

PubMed2000

Effect of host bacteria genotype on spontaneous reversions of Bacillus subtilis bacteriophage phi29 sus17 nonsense codon.

Gene 17 of Bacillus subtilis bacteriophage Phi29 is an early gene playing a role in DNA replication. Its mutant sus17(112) carries the TAA nonsense triplet at the fifth codon of the gene. We isolated and sequenced 73 spontaneous revertants producing normal-size plaques on bacteria without an informational suppressor gene. In all revertants, the TAA triplet was changed by a one-base substitution and the sequences CAA, AAA, TTA, TAC and TAT were recovered at its place. The spectrum of these mutations was markedly influenced by the genotype of the bacteria in which the revertants arose. In agreement with the results described in Escherichia coli, the ratio of transversions to transitions (CAA being the only transition acceptable) was higher in strains harboring the functional allele recA(+) than in those with recA4. Our results support the idea that also in the Gram-positive B. subtilis, the spectra of spontaneous mutations are specifically modified by an SOS function. It is assumed that the single-stranded DNA chains generated in the course of phage DNA replication might act as an inducing factor.

Bacillus Phages↗

PubMed1994

Low cytoplasmic mRNA levels of immunoglobulin kappa light chain genes containing nonsense codons correlate with inefficient splicing.

We have previously reported down-regulation of mRNA expression of some of the kappa light chain transgenes in a hybridoma derived from a secondary immune response. Of the five heavily mutated transgene copies present in that hybridoma, three included premature stop codons and were poorly represented at the mRNA level. Here we show that the nonsense mutations are the cause of the low mRNA levels. While we found no evidence that the reduction in mRNA abundance was attributable to an increased rate of cytoplasmic mRNA decay, the amount of cytoplasmic mRNA correlated with the accumulation of unspliced transcripts in the nucleus. Similar results were obtained with a chimeric immunoglobulin gene containing a premature chain termination codon in the variable gene segment. We suggest that inhibition of splicing induced by in-frame premature stop codons is an important mechanism for down-regulation of undesirable immunoglobulin transcripts.

Animals↗

PubMed2003

Nonsense mediated decay downregulates conserved alternatively spliced ABCC4 transcripts bearing nonsense codons.

Drug transporters are an important part of the defense of cells against cytotoxic agents. One major group of transporters is known as multidrug resistance associated proteins (MRP; ABCC gene family). The MRPs belong to the ATP binding cassette transporter superfamily. One family member, ABCC4 (also known as MRP4) functions as a cellular efflux pump for anti-HIV drugs, such as 9-(2-phoshoenylmethoxyethyl) adenine and azido-thymidine-monophosphate, an antiviral nucleotide, ganciclovir-monophosphate, and anti-cancer agents such as thiopurines. We isolated a ABCC4 cDNA encoding a non-functional protein, owing to an insertion, and subsequently determined the ABCC4 gene structure. This analysis revealed that the insertion was attributed to two additional exons that would be predicted to produce premature termination codons (PTC) in ABCC4. The highly similar mouse Abcc4 gene also contained these exons, which were remarkable because their size and sequence identity were much higher than the overall similarity between these genes. Further, a comparison of human, monkey and rodent ABCC4 genes revealed that these same PTC-producing exons were also highly conserved in evolution. As all the ABCC4 mRNA containing these PTC exons might produce nonsense mRNA, we further tested the hypothesis that these mRNAs were targets of nonsense-mediated mRNA decay (NMD). Protein synthesis inhibition selectively stabilized PTC containing ABCC4 transcripts in human, monkey and rodent cell lines. Moreover, the amount of PTC-containing ABCC4 transcripts was critically dependent upon protein synthesis, as removal of the inhibitor dramatically decreased expression, which correlated with the resumption of protein synthesis. These are the first studies to indicate that the highly conserved PTC exons of the ABCC4 gene may dictate its expression.

Alternative Splicing↗

PubMed2006

AUG sequences are required to sustain nonsense-codon-mediated suppression of splicing.

More than 90% of human genes are rich in intronic latent 5' splice sites whose utilization in pre-mRNA splicing would introduce in-frame stop codons into the resultant mRNAs. We have therefore hypothesized that suppression of splicing (SOS) at latent 5' splice sites regulates alternative 5' splice site selection in a way that prevents the production of toxic nonsense mRNAs and verified this idea by showing that the removal of such in-frame stop codons is sufficient to activate latent splicing. Splicing control by SOS requires recognition of the mRNA reading frame, presumably recognizing the start codon sequence. Here we show that AUG sequences are indeed essential for SOS. Although protein translation does not seem to be required for SOS, the first AUG is shown here to be necessary but not sufficient. We further show that latent splicing can be elicited upon treatment with pactamycin-a drug known to block translation by its ability to recognize an RNA fold-but not by treatment with other drugs that inhibit translation through other mechanisms. The effect of pactamycin on SOS is dependent neither on steady-state translation nor on the pioneer round of translation. This effect is found for both transfected and endogenous genes, indicating that SOS is a natural mechanism.

Animals↗

PubMed1991

CGG: an unassigned or nonsense codon in Mycoplasma capricolum.

CGG is an arginine codon in the universal genetic code. We previously reported that in Mycoplasma capricolum, a relative of Gram-positive eubacteria, codon CGG did not appear in coding frames, including termination sites, and tRNA(ArgCCG) pairing with codon CGG, was not detected. These facts suggest that CGG is a nonsense (unassigned and untranslatable) codon--i.e., not assigned to arginine or to any other amino acid. We have investigated whether CGG is really an unassigned codon by using a cell-free translation system prepared from M. capricolum. Translation of synthetic mRNA containing in-frame CGG codons does not result in "read-through" to codons beyond the CGG codons--i.e., translation ceases just before CGG. Sucrose-gradient centrifugation profiles of the reaction mixture have shown that the bulk of peptide that has been synthesized is attached to 70S ribosomes and is released upon further incubation with puromycin. The result suggests that the peptide is in the P site of ribosome in the form of peptidyl-tRNA, leaving the A site empty. When in-frame CGG codons are replaced by UAA codons in mRNA, no read-through occurs beyond UAA, just as in the case of CGG. However, the synthesized peptide is released from 70S ribosomes, presumably by release factor 1. These data suggest strongly that CGG is an unassigned codon and differs from UAA in that CGG is not used for termination.

Amino Acid Sequence↗

PubMed2000

A nonsense codon of exon 14 reduces lysyl hydroxylase mRNA and leads to aberrant RNA splicing in a patient with Ehlers-Danlos syndrome type VI.

Ehlers-Danlos syndrome type VI (EDSVI) is an autosomal recessively inherited connective tissue disease, characterized by kyphoscoliosis, muscular hypotonia and ocular manifestations. The cause of the syndrome is a deficiency in the activity of lysyl hydroxylase (LH), one of the enzymes involved in the post-translational modification of collagens. We describe here an unusual compound heterozygote British patient with EDSVI. Our investigations indicate that a maternally inherited nonsense mutation (Y511X) in exon 14 of the LH gene (PLOD1) results in a reduction of the mRNA level as well as a skipping of exon 14 sequences in the mRNA that produces a protein shortened by 38 amino acids. The transcription of the other allele of the LH gene is considerably reduced from the normal for reasons that are not yet known. As a consequence, the LH activity of the skin fibroblasts of the patient is markedly reduced.

Blotting, Southern↗

PubMed1975

Yeast super-suppressors are altered tRNAs capable of translating a nonsense codon in vitro.

tRNA isolated from two different yeast super-suppressor strains translates a known nonsense mutation in vitro, whereas tRNA from a closely related nonsuppressing strain does not. Suppression was assayed by translation of RNA isolated from an amber coat mutant of bacteriophage Qbeta (GB11) in a protein-synthesizing system derived from mouse tissue culture cells (L cells). Suppressed forms of Qbeta coat protein synthesized in vitro were quantitatively detected by a specific immunoprecipitation assay. The L-cell protein-synthesizing system also responds to E. coli suppressor tRNA. This indicates that the biochemical mechanism for nonsense suppression is very similar in yeast and E. coli. These findings also provide additional evidence that the amber codon (UAG) functions as one of the mammalian chain-terminating codons. Since the suppression assay utilizes protein-synthesizing components isolated from mammalian cells, it should prove useful in the search for mammalian nonsense suppressors.

Codon↗

PubMed1979

A streptomycin-resistant Escherichia coli mutant with ribosomes temperature-sensitive in the suppression of a nonsense codon.

Cell free extracts from a streptomycin-resistant E. coli mutant which is also temperature-sensitive for Q beta phage were studied for suppression of a nonsense mutation at various temperatures. The streptomycin-resistant ribosomes of the mutant were found to be temperature-sensitive in suppression of an amber mutation in f2 phage coat protein while retaining the ability to synthesize proteins at an elevated temperature (42 degrees C). The restriction of amber suppression at 42 degrees C is assumed to be related to an alteration in ribosomal protein S12 of the streptomycin-resistant mutant which also causes a change in its electrophoretic mobility.

Cell-Free System↗

PubMed1981