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PubMed1997

T cell receptor (TCR) mini-gene mRNA expression regulated by nonsense codons: a nuclear-associated translation-like mechanism.

Premature termination codons (PTCs) are known to decrease mRNA levels. Here, we report our investigation of the mechanism for this downregulation using the TCR-beta gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic development. We found that a mini-gene version of this gene, which contains only three TCR-beta exons, exhibited efficient downregulation in response to PTCs. This demonstrates that the full coding sequence is not necessary for appropriate regulation. Mutation of the translation start AUG and a downstream in-frame AUG that displayed similarity to the Kozak consensus sequence reversed the downregulatory response to PTCs. Thus, an AUG start codon is required to define the reading frame of a PTC. Specific suppressor tRNAs also reversed the downregulatory response, strongly implicating the involvement of a translation-like process. Remarkably, the addition of suppressor tRNAs or the inactivation of the start AUGs caused a dramatic rise in the levels of PTC-bearing transcripts in the nuclear fraction prepared by two independent methods. Collectively, our results provide evidence for a codon-based surveillance mechanism associated with the nucleus that downregulates aberrant transcripts encoding potentially toxic polypeptides from nonproductively rearranged genes.

Animals↗

PubMed1998

High prevalence of codon 213Arg-->Stop mutations of the TP53 gene in human ovarian cancer in the southwestern part of The Netherlands.

As in many human malignancies, TP53 mutations are the most common genetic alterations in malignant human ovarian tumours. An approach often used in the determination of TP53 status is immunohistochemical staining of the protein. Non-missense mutations, especially those of the null type, causing premature termination codons and resulting in truncated proteins, may often not be detectable by immunohistochemistry. Therefore, current estimates of TP53 alterations in ovarian cancer may be inaccurate. By using polymerase chain reaction-single strand conformation polymorphism analysis and sequencing techniques, we have found a high prevalence of TP53 non-missense mutations in exons 5-8 in ovarian tumour specimens from patients from the southwestern part of The Netherlands. Twenty-nine of 64 tumours showed mutations, of which 10 were non-missense mutations. The majority (9 of 10) of these non-missense mutations, including 7 nonsense mutations and 2 frameshift deletions, were null type mutations and could not be detected by immunohistochemical staining. Five of the 7 nonsense mutations were mutations at codon 213 (Arg-->Stop). The nature of the high prevalence of this nonsense mutation in our series of ovarian carcinomas remains unknown. In addition to the 9 null type mutations, a splice junction mutation was encountered. In conclusion, we have observed a high prevalence (13%) of ovarian tumours with null type mutations in exons 5-8 that did not result in immunostaining. Our data suggest that, especially in ovarian cancer, immunological assessment of TP53 is not an adequate tool to study TP53 alteration. A frequent nonsense mutation at codon 213 in 5 (8%) of 64 tumour specimens represents an important finding.

Adult↗

PubMed1981

Nonsense and frameshift mutations in beta 0-thalassemia detected in cloned beta-globin genes.

The molecular basis for deficiency of beta-globin synthesis in beta-thalassemia was investigated by gene cloning and DNA sequencing. beta-Globin genes of two patients with beta 0-thalassemia were cloned in a phage lambda vector. Both beta-genes transcribed normally in vitro. The gene of an Italian individual had a single nucleotide substitution (C leads to T) in the codon for amino acid 39 that resulted in formation of a nonsense codon. In a Turkish individual, the cloned beta-globin gene had a dinucleotide deletion in the codon for amino acid 8. This frameshift mutation produced a termination codon at the position of the new 21st codon. Mutations that lead to premature termination of beta-globin synthesis appear to be among the common causes of beta 0-thalassemia in man.

Amino Acid Sequence↗

PubMed2006

The canonical UPF1-dependent nonsense-mediated mRNA decay is inhibited in transcripts carrying a short open reading frame independent of sequence context.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs carrying premature translation termination codons. Generally, NMD is elicited if translation terminates >50-54 nucleotides (nt) upstream of an exon-exon junction. We have previously reported that human beta-globin mRNAs carrying 5'-proximal nonsense mutations (e.g., beta15) accumulate to normal levels, suggesting an exception to the "50-54-nt boundary rule." In the present report, we demonstrate that the strength of the UPF1-dependent NMD of mutant beta-globin mRNAs is specifically determined by the proximity of the nonsense codon to the initiation AUG. This conclusion is supported by a parallel effect of the short ORF size on NMD of nonsense-containing alpha-globin mRNAs. To determine whether the short-ORF effect on NMD response is conserved in heterologous transcripts, we assessed its effects on a set of beta-globin/triosephosphate isomerase (TPI) hybrid mRNAs and on the TPI mRNA. Our data support the conclusion that nonsense mutations resulting in a short ORF are able to circumvent the full activity of the canonical UPF1-dependent NMD pathway.

5' Untranslated Regions↗

PubMed1990

RNA editing of ATPase subunit 9 transcripts in Oenothera mitochondria.

The mRNA for subunit 9 of the ATPase (atp9) in the higher plant Oenothera is edited in four nucleotide positions. Three events alter genomic serine and proline codons to triplets specifying leucine. A UGA termination codon is introduced into the reading frame by modification of a CGA arginine codon. This modification shortens the polypeptide by four amino acids. Direct sequencing of PCR amplified cDNA from the total mitochondrial mRNA population gives no indication of partially edited transcripts suggesting a rapid and efficient modification of atp9 transcripts in Oenothera mitochondria.

Amino Acid Sequence↗

PubMed2001

Clinical features of a novel TIMP-3 mutation causing Sorsby's fundus dystrophy: implications for disease mechanism.

AIMS: To describe the phenotype in three family members affected by a novel mutation in the gene coding for the enzyme tissue inhibitor of metalloproteinase-3 (TIMP-3). METHODS: Three members of the same family were seen with a history of nyctalopia and visual loss due to maculopathy. Clinical features were consistent with Sorsby's fundus dystrophy. Exon 5 of the gene coding for TIMP-3 was amplified by the polymerase chain reaction, single strand conformation polymorphism analysis undertaken and exon 5 amplicons were directly sequenced. RESULTS: Onset of symptoms was in the third to fourth decade. Five of six eyes had geographic macular atrophy rather than neovascularisation as a cause for central visual loss. Peripheral retinal pigmentary disturbances were present. Scotopic ERGs were abnormal in all three. Mutation analysis showed a G-->T transversion in all three resulting in a premature termination codon, E139X, deleting most of the carboxy terminal domain of TIMP-3. CONCLUSIONS: The patients described had a form of Sorsby's fundus dystrophy which fell at the severe end of the spectrum of this disease. Postulated disease mechanisms include deposition of dimerised TIMP-3 protein.

Adult↗

PubMed2006

The NEMO mutation creating the most-upstream premature stop codon is hypomorphic because of a reinitiation of translation.

Amorphic mutations in the NF- kappa B essential modulator (NEMO) cause X-dominant incontinentia pigmenti, which is lethal in males in utero, whereas hypomorphic mutations cause X-recessive anhidrotic ectodermal dysplasia with immunodeficiency, a complex developmental disorder and life-threatening primary immunodeficiency. We characterized the NEMO mutation 110_111insC, which creates the most-upstream premature translation termination codon (at codon position 49) of any known NEMO mutation. Surprisingly, this mutation is associated with a pure immunodeficiency. We solve this paradox by showing that a Kozakian methionine codon located immediately downstream from the insertion allows the reinitiation of translation. The residual production of an NH(2)-truncated NEMO protein was sufficient for normal fetal development and for the subsequent normal development of skin appendages but was insufficient for the development of protective immune responses.

Amino Acid Sequence↗

PubMed1987

Expression of the delta 1-crystallin gene: salt-dependent alteration in the cell-free synthesis of delta-crystallin polypeptides.

We have investigated the origins of the two major size classes of delta-crystallin polypeptides (48 kDa and 50 kDa on sodium dodecyl sulfate-urea-polyacrylamide gels) in the chicken lens. Both the 48-kDa and 50-kDa polypeptides were produced by cell-free translation in a salt-dependent (Na+, K+, acetate and Cl-) pattern from mRNA derived from a cloned delta 1-crystallin cDNA. The salt-dependent alteration in the ratio of cell-free synthesis of the 48-kDa to 50-kDa delta-crystallin polypeptides was greatly enhanced by capping of the delta 1 mRNA. Translation of the delta 1 mRNA containing a premature termination codon suggested that the larger delta-crystallin band contains two polypeptides which differ in their N-terminal one-third. In vitro transcription/translation analysis of several mutant delta 1 cDNA clones and immunoblot analysis of authentic delta-crystallin with antisera raised to peptides contained in delta-crystallin showed that neither alternative initiation at two in-phase AUG codons nor alternative termination at sites on the delta 1 mRNA are responsible for generating the two sizes of the delta-crystallin polypeptides. Taken together our data suggest (but do not prove) that delta-crystallin heterogeneity is generated by co-translational modification of the primary polypeptide encoded in the delta 1-crystallin gene.

Acetates↗

PubMed1995

Transcription termination of the streptokinase gene of Streptococcus equisimilis H46A: bidirectionality and efficiency in homologous and heterologous hosts.

In Streptococcus equisimilis H46A, a hypersymmetrical transcription terminator with bidirectional activity was localized between the translational termination codons of the streptokinase gene, skc, and the rel-orf1 genes. These two transcription units are oriented towards each other, and under normal conditions the skc mRNA level exceeds that of the rel-orf1 genes by a factor of at least 1000. Reporter vectors based on the promoterless cat gene were constructed by transcriptional fusion of skc to cat, such that the region between the two genes contained the terminator in skc orientation or in rel-orf1 orientation. Additionally, skc and cat were fused directly, with deletion of the terminator. The reporter vectors were designed to be capable of being studied either as multicopy plasmids in Escherichia coli or in single copy following integration, via skc, into the S. equisimilis chromosome. Chloramphenicol acetyl transferase (CAT) activity assays in conjunction with determination of chloramphenicol resistance levels and Northern hybridization analysis showed that the terminator is active in either host and orientation. However, termination efficiency was host dependent, with high terminator strength being observed in the homologous streptococcal background and appreciable readthrough occurring in E. coli. The extent of transcriptional readthrough was dependent upon terminator orientation, with termination being more efficient in rel-orf1 polarity. The results suggest that, in S. equisimilis, transcription of both skc and rel-orf1 is efficiently terminated by a common signal, and that these genes are largely protected from convergent transcription, which otherwise would seem to be particularly detrimental to the weakly expressed rel-orf1 genes.

Base Sequence↗

PubMed1994

tRNA structure and ribosomal function. I. tRNA nucleotide 27-43 mutations enhance first position wobble.

Transfer RNA su7 G36 is a derivative of tRNA(Trp) with a 3'GUC anticodon complementary to the glutamine codon CAG. This tRNA requires a normally forbidden G-U wobble at the first codon position to suppress a UAG (amber) termination codon. Measurement of amber suppression by mutated su7 G36 tRNAs and correction for tRNA levels and aminoacylation allowed calculation of KUAG, a linearized index of in vivo ribosomal function. Following saturating mutagenesis of the anticodon arm of su7 G36, screening for UAG suppression using a lacZ reporter yielded tRNAs with up to 40-fold increased first position G-U wobble, judged from KUAG. The parental anticodon helix has minimized this type of miscoding, and virtually all changes in the top base-pair of the anticodon helix, nucleotides (nt) 27-43, increased the error. Thus, misincorporation of amino acids due to aberrant first position wobble is apparently prevented by normal tRNA structure, which is specifically altered by substitution at nt 27-43, the top base-pair of the anticodon helix. All 16 permutations of nt 27-43, the hotspot for increased wobble, were subsequently constructed and compared. Comparison of values for tRNA coding function, tRNA level, and aminoacylation for the 16 suggest that a tRNA conformational change, specifically involving both nt 27-43, differentially affects all these tRNA functions. This conformational alteration, which presumably occurs normally on the ribosome, appears more complex than simple breakage of the normal 27-43 base-pair. We suggest that the change is in the angle and/or flexibility of the tRNA L-shape. Among these 16 tRNAs, efficient wobble is strongly and inversely correlated with good aminoacylation and high tRNA levels; this quality may have been selected. Constraints on the sequences of natural tRNAs suggest that nt 27-43 have effects on function in many tRNAs.

Anticodon↗

PubMed1993

High-sequence conservation among the United States bluetongue viruses cognate M2 genes which encode the nonstructural NS1 tubule protein.

Full-length cDNA copies of the M2 gene of BTV-2, -11, and -13 serotypes obtained by a modified polymerase chain reaction (Clamp-R) were cloned into pUC19 plasmid. The entire nucleotide sequences of each M2 gene were determined and compared with BTV-10 and BTV-17, thus completing the sequencing of these cognate M2 gene segments from all five U.S. BTV serotypes. Each M2 segment contained 1769 nucleotides, a single open reading frame (ORF) with an initiation codon at nucleotides 35-37, and a termination codon at nucleotides 1691-1693. This ORF can encode the 552-amino-acid NS1 protein (64 KDa) which has an isoelectric point of 7. Analyses of the nucleotide and deduced amino acid sequences of the five U.S. BTV serotypes indicated that the most recently isolated BTV-2 serotype was more distantly related than BTV-10, -11, -13, or -17. Analyses of the evolutionary relatedness of the cognate M2 genes by codon positions indicate that the rate of mismatch accumulations in the first and second base codon positions are less than 4%. However, the mismatch accumulations in the third base codon position are quite evident (23%) when BTV-2 serotype was compared with the other U.S. BTV serotypes. This suggests that BTV-2 has separated from the other four U.S. serotypes long before they themselves diverged. These data also indicate that the five U.S. BTV serotypes were apparently derived from two distinct gene pools that reflected geographic distribution in North America.

Base Sequence↗

PubMed1995

Sequence analyses and antigenic epitope mapping of the putative RNA-directed RNA polymerase of five U.S. bluetongue viruses.

We determined the complete nucleotide sequences of the cognate L1 double-stranded RNA segments of bluetongue virus (BTV) serotypes 2, 11, 13, and 17, which encode the putative RNA-directed RNA polymerase VP1. Each L1 gene contained 3944 nucleotides and was 10 bases shorter than the previously reported L1 gene of BTV 10. A single open reading frame which could encode the reported VP1 protein, 1302 amino acids in size, began with an initiation codon at nucleotides 12-14 and a termination codon at nucleotides 3918-3920. Analyses of the nucleotides of L1 genes and the deduced amino acid sequences of VP1 proteins of the five U.S. BTV serotypes indicated that the most recently isolated BTV-2 serotype from Florida was more distantly related than BTV-10, 11, 13, and 17, which were isolated primarily in the western U.S.A. The results are consistent with our hypothesis that BTVs-10, -11, -13, and -17 are derived from a single and common gene pool, and that BTV-2 belongs to a second, distinct gene pool. These genetic distinctions also reflected well with the known geographic distribution of the five U.S. BTV serotypes in North America. This putative RNA-directed RNA polymerase (149 KDa) was a basic protein, and the deduced amino acid sequences of the VP1 proteins contained seven highly conserved hydrophobic domains and many other sequence motifs which were also found in other known RNA polymerases. Four immunodominant but linear antigenic epitopes conserved among the VP1 of five U.S. BTVs were also been identified and mapped using monospecific oligoclonal antibodies.

Amino Acid Sequence↗

PubMed1988

Nucleotide homology and organization of chlorocatechol oxidation genes of plasmids pJP4 and pAC27.

The 2,4-dichlorophenoxyacetate (2,4-D) catabolic plasmid pJP4 of Alcaligenes eutrophus JMP134 contains two sets of nonidentical chlorocatechol oxidation gene sequences physically separated by a 7 kb DNA region. We determined the nucleotide sequence of the 1.6 kb HindIII fragment containing the known genes tfdC and tfdD (Don et al. 1985) which encode pyrocatechase and cycloisomerase, respectively. The 1.3 kb BglII-HindIII segment of recombinant plasmid pDC25 containing at least three chlorocatechol (clc) oxidation genes of the pAC27 plasmid in Pseudomonas putida AC867 (Ghosal et al. 1985a; Frantz and Chakrabarty 1986), was also sequenced. When the tfdC gene of the pJP4 plasmid was compared with gene clcA of plasmid pAC27, which encodes the chlorocatechol specific pyrocatechase (pyrocatechase II), the two genes showed 63% nucleotide sequence homology with 60% homology in their amino acid sequences. In both plasmid pJP4 and pAC27, the two genes encoding the pyrocatechase and the cycloisomerase showed a 4 bp overlap spanning the initiation codon of the cycloisomerase gene and the termination codon of the pyrocatechase gene. The sizes of the polypeptides encoded by the isofunctional genes tfdC and clcA are very similar and thus reflect their functional homology.

Alcaligenes↗

PubMed1990

Nucleotide sequence and characterization of the 39K gene region of Autographa californica nuclear polyhedrosis virus.

The complete nucleotide sequence of the Pstl-K fragment of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome was determined. This region of the genome contains the delayed early 39K gene and V-ubi, a viral protein with homology to ubiquitine. In addition, Pstl-K potentially encodes five other proteins. Hybrid-select translation mapped nine viral-specific proteins to this fragment. Three proteins were observed with both early and late RNA; two of them comigrated with proteins directed by transcripts made from the 39K open reading frame (ORF). One protein which was only seen with hybrid-selected late RNA comigrated with ubiquitin. A correction of previously published sequence data indicates that there is an additional AUG codon upstream of the 39K ORF. This AUG codon is located 8 nucleotides downstream of the early transcription initiation site. The methionine codon is followed by codons for three amino acids and a termination codon. The 39K ORF is located 102 nucleotides downstream of the minicistron. In vitro transcription-translation experiments confirmed that the downstream AUG serves as the initiation codon for the 39K ORF. Radioimmunoprecipitation experiments indicated that the Pstl-K-encoded 39K protein reacted with antiserum raised against a previously described nuclear matrix-associated 39K protein.

Amino Acid Sequence↗

PubMed1991

Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome.

We have recently described the cloning of a portion of the hepatitis E virus (HEV) and confirmed its etiologic association with enterically transmitted (waterborne, epidemic) non-A, non-B hepatitis. The virus consists of a single-stranded, positive-sense RNA genome of approximately 7.5 kb, with a polyadenylated 3' end. We now report on the cloning and nucleotide sequencing of an overlapping, contiguous set of cDNA clones representing the entire genome of the HEV Burma strain [HEV(B)]. The largest open reading frame extends approximately 5 kb from the 5' end and contains the RNA-directed RNA polymerase and nucleoside triphosphate binding motifs. The second major open reading frame (ORF2) begins 37 bp downstream of the first and extends approximately 2 kb to the termination codon present 65 bp from the 3' terminal stretch of poly(A) residues. ORF2 contains a consensus signal peptide sequence at its amino terminus and a capsid-like region with a high content of basic amino acids similar to that seen with other virus capsid proteins. A third open reading frame partially overlaps the first and second and encompasses only 369 bp. In addition to the 7.5-kb full-length genomic transcript, two subgenomic polyadenylated messages of approximately 3.7 and 2.0 kb were detected in infected liver using a probe from the 3' third of the genome. The genomic organization of the virus is consistent with the 5' end encoding nonstructural and the 3' end encoding the viral structural gene(s). The expression strategy of the virus involves the use of three different open reading frames and at least three different transcripts. HEV was previously determined to be a nonenveloped particle with a diameter of 27-34 nm. These findings on the genetic organization and expression strategy of HEV suggest that it is the prototype human pathogen for a new class of RNA virus or perhaps a separate genus within the Caliciviridae family.

Amino Acid Sequence↗

PubMed1994

Analyses and conservation of sequences among the cognate L3 segments of the five United States bluetongue viruses.

We determined the complete nucleotide sequences of the cognate L3 double-stranded RNA (ds-RNA) segments of bluetongue virus (BTV) serotypes 2, 11, and 13 encoding the major viral inner capsid protein, VP3. Each cognate L3 segment was 2772 nucleotides long and contained a single open reading frame (ORF) with an initiation codon at nucleotides #18-20 and a termination codon at nucleotides #2721-2723. This ORF can encode the 901-amino acid VP3 protein (103 kDa) with a calculated isoelectric point of 6. Phylogenetic analyses using both the nucleotide and the deduced amino acid sequences of the L3 cognate gene of the five US BTV serotypes indicated that the BTV-2 serotype recently isolated in Florida was more distantly related than BTV-10, 11, 13 or 17. The five US BTV serotypes were derived apparently from two distinct gene pools, findings consistent with their current geographic distribution in North America.

Amino Acid Sequence↗

PubMed2006

Suppression of galactosylceramidase (GALC) expression in the twitcher mouse model of globoid cell leukodystrophy (GLD) is caused by nonsense-mediated mRNA decay (NMD).

The twitcher mouse is a pathologically and enzymatically authentic model of globoid cell leukodystrophy (GLD, Krabbe disease) that has been widely used for the evaluation of potential therapeutic approaches. This naturally occurring mouse model contains a premature stop codon (W339X) in the galactosylceramidase (GALC) gene that abolishes enzymatic activity. Using either immunocytochemical approaches or Western blot methodology, we have been unable to detect the truncated form of GALC expected to be produced in these animals. Nonsense-mediated mRNA decay (NMD) is a cellular protection mechanism that degrades newly synthesized transcripts containing a premature termination codon (PTC). Since the naturally occurring mutation in the twitcher mouse introduces a PTC, we hypothesized that NMD might affect the degradation of GALC mRNA in these animals. Consistent with this hypothesis, we determined that the amount of GALC transcript was inversely proportional to the number of twitcher containing alleles. Similar reductions in GALC mRNA were detected in a twitcher-derived Schwann cell line (TwS1) when compared to wild-type Schwann cells (IMS32). Anisomycin, emetine and puromycin, inhibitors of NMD, effectively increased the level of GALC transcript in the TwS1 cells providing further support for nonsense-mediated mRNA decay being the mechanism by which no GALC protein is detected in these animals. Understanding the mechanistic differences between the lack of enzymatic activity in the twitcher model and that observed with the missense mutations that cause human disease yields not only novel therapeutic insights but also highlights the need for additional animal models.

Amidines↗

PubMed1996

Nonsense mutations inhibit RNA splicing in a cell-free system: recognition of mutant codon is independent of protein synthesis.

Mutations resulting in premature termination codons reduce the corresponding mRNA levels. We describe a cell-free system in which depletion of the mutant immunoglobulin kappa mRNA pool correlates with inefficient splicing and not with RNA decay. Splicing deficiency does not depend on the sequence surrounding the in-frame nonsense codon and can be partially corrected by mutating the methionine initiation codon. Despite the apparent link between translation and low mutant mRNA levels, inefficient splicing is not dependent on protein synthesis. Abnormal splicing of mutant immunoglobulin RNA is observed with B-cell but not with HeLa or T-cell extracts. A nonsense mutant beta-globin RNA is normally spliced by B-cell extract. We propose that the phenomenon exhibits tissue and gene specificity.

Animals↗