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PubMed1986

Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy.

Effects on translation in vivo by modification deficiencies for 2-methylthio-N6-isopentenyladenosine (ms2i6A) (Escherichia coli) or 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) (Salmonella typhimurium) in tRNA were studied in mutant strains. These hypermodified nucleosides are present on the 3' side of the anticodon (position 37) in tRNA reading codons starting with uridine. In E. coli, translational error caused by tRNA was strongly reduced in the case of third-position misreading of a tryptophan codon (UGG) in a particular codon context but was not affected in the case of first-position misreading of an arginine codon (CGU) in another codon context. Misreading of UGA nonsense codons at two different positions was codon context dependent. The efficiencies of some tRNA nonsense suppressors were decreased in a tRNA-dependent manner. Suppressor tRNA which lacks ms2i6A-ms2io6A becomes more sensitive to codon context. Our results therefore indicate that, besides improving translational efficiency, ms2i6A37 and ms2io6A37 modifications in tRNA are also involved in decreasing the intrinsic codon reading context sensitivity of tRNA. Possible consequences for regulation of gene expression are discussed.

Anticodon↗

PubMed2000

Forced retroevolution of an RNA bacteriophage.

The operator hairpin ahead of the replicase gene in RNA bacteriophage MS2 contains overlapping signals for binding the coat protein and ribosomes. Coat protein binding inhibits further translation of the gene and forms the first step in capsid formation. The hairpin sequence was partially randomized to assess the importance of this structure element for the bacteriophage and to monitor alternative solutions that would evolve on the passaging of mutant phages. The evolutionary reconstruction of the operator failed in the majority of mutants. Instead, a poor imitation developed containing only some of the recognition signals for the coat protein. Three mutants were of particular interest in that they contained double nonsense codons in the lysis reading frame that runs through the operator hairpin. The simultaneous reversion of two stop codons into sense codons has a very low probability of occurring. Therefore the phage solved the problem by deleting the nonsense signals and, in fact, the complete operator, except for the initiation codon of the replicase gene. Several revertants were isolated with activities ranging from 1% to 20% of wild type. The operator, long thought to be a critical regulator, now appears to be a dispensable element. In addition, the results indicate how RNA viruses can be forced to step back to an attenuated form.

Base Sequence↗

PubMed2000

Nonsense mutations in the human beta-globin gene lead to unexpected levels of cytoplasmic mRNA accumulation.

Generally, nonsense codons 50 bp or more upstream of the 3'-most intron of the human beta-globin gene reduce mRNA abundance. In contrast, dominantly inherited beta-thalassemia is frequently associated with nonsense mutations in the last exon. In this work, murine erythroleukemia (MEL) cells were stably transfected with human beta-globin genes mutated within each of the 3 exons, namely at codons 15 (TGG-->TGA), 39 (C-->T), or 127 (C-->T). Primer extension analysis after erythroid differentiation induction showed codon 127 (C-->T) mRNA accumulated in the cytoplasm at approximately 20% of the normal mRNA level. Codon 39 (C-->T) mutation did not result in significant mRNA accumulation. Unexpectedly, codon 15 (TGG-->TGA) mRNA accumulated at approximately 90%. Concordant results were obtained when reticulocyte mRNA from 2 carriers for this mutation was studied. High mRNA accumulation of codon 15 nonsense-mutated gene was revealed to be independent of the type of nonsense mutation and the genomic background in which this mutation occurs. To investigate the effects of other nonsense mutations located in the first exon on the mRNA level, nonsense mutations at codons 5, 17, and 26 were also cloned and stably transfected into MEL cells. After erythroid differentiation induction, mRNAs with a mutation at codon 5 or 17 were detected at high levels, whereas the mutation at codon 26 led to low mRNA levels. These findings suggest that nonsense-mediated mRNA decay is not exclusively dependent on the localization of mutations relative to the 3'-most intron. Other factors may also contribute to determine the cytoplasmic nonsense-mutated mRNA level in erythroid cells. (Blood. 2000;96:2895-2901)

Animals↗

PubMed1997

Structural alterations far from the anticodon of the tRNAProGGG of Salmonella typhimurium induce +1 frameshifting at the peptidyl-site.

A total of 12 Salmonella typhimurium mutants were selected with mutations in the minor tRNAProGGG which suppress a +1 frameshift mutation in the hisD gene. This tRNA normally has 1-methylguanosine (m1G37) next to and 3' of the anticodon (position 37). Since the presence of m1G37 prevents frameshifting, some of the +1 frameshift suppressor derivatives of tRNAProGGG had alterations in the primary sequence abolishing the formation of m1G37. However, several of the mutant tRNAProGGG species had a normal level of m1G37 and a normal-sized anticodon loop, showing that neither m1G37 deficiency, nor an oversized anticodon loop, is a prerequisite for +1 frameshifting. Moreover, base substitutions far from the anticodon, e.g. in the acceptor stem, DHU-loop and stem, and at the top of the anticodon stem, promoted +1 frameshifting. When the frameshifting site (CCC-Uaa; CCC is in the zero frame and a +1 frameshift moves the ribosome to the CC-U codon) is overlapped by a nonsense codon (UAA), the efficiency of frameshifting decreased when release factor 1 was over-expressed and increased at an elevated temperature in a mutant with a temperature-sensitive release factor 1. The frameshifting site (CCC-Uac) was also overlapped with the sense codon UAC, which is decoded by a tRNA species having a 2-methylthio-cis ribozeatin (ms2io6A) at position 37. Mutations in the miaA gene affect the formation of this modified nucleoside and result in an A instead of ms2io6A37 in the tRNA. Such an undermodified tRNA is very inefficient in translation and the efficiency of frameshifting increased in a miaA1 mutant. These results suggest that the frameshifting event occurs at the P-site, since the efficiency of frameshifting was sensitive to the decoding activity of the overlapping codon. We conclude that tRNA with mutations far from the anticodon, with a normal-sized anticodon loop and having m1G37 induce +1 frameshifting at the P-site.

Alcohol Oxidoreductases↗

PubMedDate not supplied

Nonsense-mediated mRNA decay in barley mutants allows the cloning of mutated genes by a microarray approach.

We have previously described a microarray approach to identify and clone genes from mutants of higher organisms. In the method cDNA of two mutants with similar phenotype are competitively hybridized to DNA clones arrayed on a glass slide. Clones corresponding to an mRNA that is not expressed in one of the strains due to a mutation will be specifically highlighted in the hybridization, which provides a possibility to identify and eventually clone the mutated gene. The approach is dependent on mutations that affect the amount of mRNA. Nonsense mutations, which prematurely terminate translation, can be such mutations as a surveillance system known as nonsense-mediated decay (NMD) has been developed by organisms to reduce the abundance of mRNA with nonsense codons. In the present study, we have analysed the barley (Hordeum vulgare L.) magnesium chelatase mutants xantha-f26, xantha-f27 and xantha-f40 in order to investigate the presence of NMD in barley, as well as the importance of the position of the stop codon for NMD. Both nonsense-mutants xantha-f27 and xantha-f40, but not the missense mutant xantha-f26, showed NMD. This was not expected for xantha-f27 as its mutation is in the last exon of the gene. We conclude the NMD expands the number of mutants that can be used for gene cloning by our described microarray approach.

Cloning, Molecular↗

PubMed1998

Molecular study and prenatal diagnosis of alpha- and beta-thalassemias in Chinese.

Thalassemia is one of the most common single gene diseases worldwide. Populations in southern China and Taiwan have high prevalence rates of alpha- and beta-thalassemias. This review summarizes the current status of molecular studies, carrier screening, and prenatal diagnosis of thalassemia in Chinese. There are three genotypes of alpha-thalassemia 1 and at least six of alpha-thalassemia 2 in Chinese. For alpha-thalassemia 1, the South-East Asian deletion is the most common, followed by the Thai then Philippino deletions. For alpha-thalassemia 2, the rightward deletion is the most common, followed by the leftward deletion, and the nondeletional defects Hb Constant Spring and Hb Quong Sze. Twenty-eight different beta-thalassemia mutations have been reported. Four mutations, IVS-II-654 (C-->T), codons 41/42 frameshift (-TCTT), and nonsense codons 17 (A-->T) and -28 (A-->G), account for more than 90% of mutant alleles. For detection of alpha-thalassemia, polymerase chain reaction-related techniques are mainly used. Southern blot hybridization is still useful, especially for prenatal diagnosis. For detection of beta-thalassemia mutations, analysis of amplification-created restriction sites and reverse dot blot hybridization have been extensively used. In Taiwan, a national screening program incorporating hematological and molecular biological methods for thalassemia detection in pregnant women has been in progress for 5 years. Prenatal diagnosis has been performed in more than 1,800 pregnancies, including 1,500 cases at risk for homozygous alpha-thalassemia 1 and 300 for beta-thalassemia major, resulting in early prenatal diagnosis and termination of pregnancies affected with homozygous alpha-thalassemia 1 and an approximately 70% decrease in the number of newborns affected with beta-thalassemia major. In mainland China, only one large-scale screening program is in place. Characterization of undefined alleles, a higher awareness of the disease among physicians and the general public, and improvement of the service network will be important for early prenatal diagnosis and prevention of the disease in the future.

China↗

PubMed2002

Mutational eidence for a functional connection between two domains of 23S rRNA in translation termination.

Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.

Codon, Nonsense↗

PubMed1994

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

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. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.

Animals↗

PubMed1999

An internal open reading frame triggers nonsense-mediated decay of the yeast SPT10 mRNA.

Yeast cells containing a temperature-sensitive mutation in the PRT1 gene were found to selectively stabilize mRNAs harboring early nonsense codons. The similarities between the mRNA decay phenotypes of prt1-1 cells and those lacking the nonsense-mediated mRNA decay (NMD) factor Upf1p led us to determine whether both types of mutations cause the accumulation of the same mRNAs. Differential display analysis and mRNA half-life measurements demonstrated that the HHF2 mRNA increased in abundance in prt1-1 and upf1Delta cells, but did not manifest a change in decay rate. In both mutant strains this increase was attributable to stabilization of the SPT10 transcript, an mRNA encoding a transcriptional regulator of HHF2. Analyses of chimeric mRNAs used to identify the cis-acting basis for NMD of the SPT10 mRNA indicated that ribosomes scan beyond its initiator AUG and initiate at the next downstream AUG, resulting in premature translation termination. By searching a yeast database for transcripts with sequence features similar to those of the SPT10 mRNA, other transcripts that decay by the NMD pathway were identified. Our results demonstrate that mRNAs undergoing leaky scanning are a new class of endogenous NMD substrate, and suggest the existence of a novel cellular regulatory circuit.

5' Untranslated Regions↗

PubMed1994

Codon context and protein synthesis: enhancements of the genetic code.

The sequence around stop codons strongly affects termination efficiency and the probability of alternative events to termination such as frameshifting and stop codon readthrough. Where tRNA suppressors of nonsense codons are present, both the efficiency of suppression and of the termination process appear to be affected by stop codon context. Since context can affect suppressor tRNA function directly, an influence on sense codon translation or suppression might be expected, but has not yet been clearly demonstrated. Statistical analyses of coding sequences indicate non-random contexts for both stop and sense codons, and non-random occurrence of codon pairs. Highly expressed genes show clear preferences among stop codons and their contexts, whereas inefficient stop signals are exploited in a variety of recoding processes.

Codon↗

PubMed2002

A bacteriophage reagent for Salmonella: molecular studies on Felix 01.

Felix 01 (F01) is a bacteriophage originally isolated by Felix and Callow which lyses almost all Salmonella strains and has been widely used as a diagnostic test for this genus. Molecular information about this phage is entirely lacking. In the present study, the DNA of the phage was found to be a double-stranded linear molecule of about 80 kb. 11.5 kb has been sequenced and in this region A + T content is 60%. There are relatively few restriction endonuclease cleavage sites in the native genome and clones show this is due to their absence rather than modification. A restriction map of the genome has been constructed. The ends of the molecule cannot be ligated although they contain 5' phosphates. At least 60% of the genome must encode proteins. In the sequenced portion, many open reading frames exist and these are tightly packed together. These have been examined for homology to published proteins but only 1 to 17 shows similarity to known proteins. F01 is therefore the prototype of a new phage family. On the basis of restriction sites, codon usage and the distribution of nonsense codons in the unused reading frames, a strong case can be made for natural selection that reacts to mRNA structure and function.

Base Sequence↗

PubMed1978

Nucleotide sequence at the 5' extremity of tobacco-mosaic-virus RNA. 2. The coding region (nucleotides 69-236).

In the preceding paper it was shown that the first A-U-G codon in tobacco mosaic virus RNA is separated from the 5' terminus by a sequence of 68 nucleotides devoid of internal guanosine residues. In this paper we present the sequence of 165 residues immediately following the first potential initiation codon. The characterized sequence contains four nonsense codons but none are in phase with the prospective initiation codon. Several lines of evidence, including direct characterization of the portion of the RNA molecule which binds to and is protected by the ribosome in the course of initiation, all support the idea that the A-U-G at position 69-71 is a functional initiation signal for viral protein synthesis.

Base Sequence↗

PubMed1998

PAX6 mutations reviewed.

Mutations in PAX6 are responsible for human aniridia and have also been found in patients with Peter's anomaly, with congenital cataracts, with autosomal dominant keratitis, and with isolated foveal hypoplasia. No locus other than chromosome 11p13 has been implicated in aniridia, and PAX6 is clearly the major, if not only, gene responsible. Twenty-eight percent of identified PAX6 mutations are C-T changes at CpG dinucleotides, 20% are splicing errors, and more than 30% are deletion or insertion events. There is a noticeably elevated level of mutation in the paired domain compared with the rest of the gene. Increased mutation in the homeodomain is accounted for by the hypermutable CpG dinucleotide in codon 240. Very nearly all mutations appear to cause loss of function of the mutant allele, and more than 80% of exonic substitutions result in nonsense codons. In a gene with such extraordinarily high sequence conservation throughout evolution, there are presumed undiscovered missense mutations, these are hypothesized to exist in as-yet unidentified phenotypes.

Aniridia↗

PubMed2006

First Spanish case of thalassemia major due to a compound heterozygosity for the IVS-II-848 (C --> A) and codon 39 (C --> T) mutations of the beta-globin gene.

This report describes the first case in Spain of a severe form of beta-thalassemia (thal) due to a compound heterozygosity for the IVS-II-848 (C --> A) and the nonsense codon 39 (C --> T) mutations. Five members of a family from Cadiz (southern Spain) were studied. The proband was an 8-year-old girl diagnosed as anemic at the age of 13 months. Her father had the codon 39 (C --> T) mutation and her mother the C --> A change at nucleotide (nt) 848 of IVS-II. Haplotype analysis showed that the proband was a compound heterozygote for haplotypes I [+ --> + +] and VII [+ --> +]. This is the first description in Spain of the IVS-II-848 (C --> A) mutation. It appears, from restriction fragment length polymorphism (RFLP) analysis, that this mutation has a different origin in the various populations, where it was found. This observation shows that in this case the association of a beta(0)- and a beta(+)-thal mutation does not lead to a thalassemia intermedia but to a severe thalassemia with very low hemoglobin (Hb) levels. From a therapeutic point of view, early introduction of a transfusion regimen may improve the clinical picture of these children, allowing for better development and growth.

Adolescent↗

PubMed2006

Variation in retinitis pigmentosa-11 (PRPF31 or RP11) gene expression between symptomatic and asymptomatic patients with dominant RP11 mutations.

Dominant mutations in the mRNA splicing factor gene PRPF31 (RP11) cause retinitis pigmentosa with reduced penetrance. We studied the expression of RP11 in lymphoblast cell lines from 10 patients, including three who were clinically asymptomatic, with six distinct RP11 mutations. Five of the six mutations were characterized and all five created premature nonsense codons or eliminated the normal initiation codon. Semiquantitative RT-PCR indicated that an average of only 17% of the RP11 mRNA was derived from the mutant allele, likely because the mutant mRNA transcripts were degraded by nonsense-mediated decay. Gene expression levels were measured by Affymetrix and CodeLink microarrays and, for RP11 transcripts, also by real-time PCR. Combined wild-type-plus-mutant RP11 mRNA expression from symptomatic patients was 52 to 77% of that in controls (p < or = 0.0005). Clinically asymptomatic carriers had levels of RP11 mRNA similar to controls and 29-42% higher than in clinically affected patients (0.0001<p<0.05, varying according to measurement technique). Expression levels of seven housekeeping genes (4-15 exons each) and 11 single-exon histone genes showed no substantial differences between affected patients with RP11 mutations and controls. Our results indicate that penetrance of dominant RP11 mutations correlates with the expression level of the remaining wild-type RP11 allele. Because RP11 mutations are apparently null alleles and because nonpenetrance correlates with high wild-type allele expression, the phenotypic effect of RP11 mutations is likely due to haploinsufficiency. The similar mRNA expression levels from genes with and without introns suggest that there is no generalized RNA splicing abnormality in RP11 patients.

Alleles↗

PubMed2005

Downregulation of eRF1 by RNA interference increases mis-acylated tRNA suppression efficiency in human cells.

The site-specific incorporation of non-natural amino acids into proteins by nonsense suppression has been widely used to investigate protein structure and function. Usually this technique exhibits low incorporation efficiencies of non-natural amino acids into proteins. We describe for the first time an approach for achieving an increased level of nonsense codon suppression with synthetic suppressor tRNAs in cultured human cells. We find that the intracellular concentration of the eukaryotic release factor 1 (eRF1) is a critical parameter influencing the efficiency of amino acid incorporation by nonsense suppression. Using RNA interference we were able to lower eRF1 gene expression significantly. We achieved a five times higher level of amino acid incorporation as compared with non-treated control cells, as demonstrated by enhanced green fluorescent protein (EGFP) fluorescence recovery after importing a mutated reporter mRNA together with an artificial amber suppressor tRNA.

Amino Acids↗

PubMed2001

A double mutation in a patient with X-linked myotubular myopathy.

In this report a double mutation was identified in a patient with X-linked myotubular myopathy. The mutations present in the patient were a C-->T substitution of nucleotide 163, which led to an Arg 55 stop codon (nonsense mutation), and an "A" insertion at nucleotide 440, which caused a shift of the reading frame and a premature stop at codon 153 (frameshift mutation). The nonsense mutation was heterozygously present in the mother but not identified in the father or in normal controls. The frameshift mutation was not identified in either parent or normal controls (de novo mutation). These mutations are predicted to truncate the myotubularin protein.

Codon, Nonsense↗

PubMed1999

A premature termination codon within an alternative exon affecting only the metabolism of transcripts that retain this exon.

Protein 4.1 pre-mRNA splicing is regulated in tissue- and development-specific manners. Exon 16, which encodes the N-terminal region of the spectrin/actin-binding domain, is one of the alternatively spliced sequence motifs. It is present in late differentiated erythroid cells but absent from early erythroblasts and from lymphoid cells. We describe a single nucleotide deletion of the erythroid protein 4.1 gene associated with hereditary elliptocytosis. The deletion located in exon 16 leads to a frameshift and a premature termination codon within the same exon. In an effort to examine the premature stop codon effect in relationship with exon 16 alternative splicing, we analyzed erythroid and lymphoid protein 4.1 mRNAs using the mutation and a linked downstream polymorphism as markers. We found that the premature stop codon does not affect the tissue-specific alternative splicing among the two cell types analyzed and that the resulting alteration of mRNA metabolism correlates with the retention of exon 16 in reticulocytes. Conversely, skipping of exon 16 in lymphoid cells converts the mutant mRNA to a normal lymphoid-specific mRNA isoform, hence bypassing the nonsense codon. Consistent with data obtained on constitutive nonsense exons, our observations argue in favor of a stop codon recognition mechanism that occurs after the regulated splicing status of the nonsense exon has been achieved.

Alternative Splicing↗