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PubMed2003

An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3'-->5' degradation.

Eukaryotic mRNAs containing premature termination codons are subjected to accelerated turnover, known as nonsense-mediated decay (NMD). Recognition of translation termination events as premature requires a surveillance complex, which includes the RNA helicase Upf1p. In Saccharomyces cerevisiae, NMD provokes rapid decapping followed by 5'-->3' exonucleolytic decay. Here we report an alternative, decapping-independent NMD pathway involving deadenylation and subsequent 3'-->5' exonucleolytic decay. Accelerated turnover via this pathway required Upf1p and was blocked by the translation inhibitor cycloheximide. Degradation of the deadenylated mRNA required the Rrp4p and Ski7p components of the cytoplasmic exosome complex, as well as the putative RNA helicase Ski2p. We conclude that recognition of NMD substrates by the Upf surveillance complex can target mRNAs to rapid deadenylation and exosome-mediated degradation.

3' Untranslated Regions↗

PubMed1999

Interaction between a mutant release factor one and P-site peptidyl-tRNA is influenced by the identity of the two bases downstream of the stop codon UAG.

Termination efficiency of a mutant form of RF (release facor) 1, as compared to the wild-type enzyme, is influenced by the P-site peptidyl-tRNA if the termination signal is UAGA. This effect is weaker at the stronger termination signal UAGU. Similarly, low efficiency of the mutant RF1, together with certain peptidyl-tRNAs, can be increased by changing the second base of the 3'-flanking codon from C to G. The data suggest that the mutant RF1 interacts with the P-site peptidyl-tRNA in conjunction with the context at the 3'-side of the termination codon.

Amino Acid Substitution↗

PubMed2026

Active Site Assembly by SMG5 as a Mechanism for SMG6 Endonuclease Licencing in Nonsense-mediated mRNA Decay.

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway that eliminates transcripts containing premature termination codons (PTCs). Substantial progress has been made in defining the transcript features that mark aberrant translation termination for NMD activation, yet key mechanistic steps remain incompletely understood - including how recruitment of the central NMD factor UPF1 is coupled to the downstream effector phase in which targeted mRNAs are nucleolytically degraded. In metazoans, NMD employs an endonucleolytic route mediated by SMG6, a PIN-domain nuclease, alongside SMG5 and SMG7, which act downstream of PTC recognition. SMG5 has recently been proposed to licence SMG6 activity, yet the molecular basis of this licencing has remained elusive. Here, we combine AlphaFold structural predictions with biochemical assays to investigate interactions among human SMG5, SMG6, and SMG7. Structural models predict a high-confidence interface between SMG5 and SMG6 PIN domains that forms a composite active site: a conserved SMG5 aspartate (D893) complements the SMG6 acidic triad to reinstate the canonical tetrad required for PIN-domain catalysis. In vitro, SMG6 alone exhibits weak endonucleolytic activity, which is enhanced ∼10-fold by the SMG5 PIN domain. Mutational analyses confirm that conserved residues from both proteins are essential for this composite configuration. Our findings reveal that the SMG5 PIN domain, previously considered catalytically inert, plays a critical role in activating SMG6 by completing its active site. This work provides mechanistic insight into the SMG5-dependent licencing step and uncovers a composite PIN nuclease architecture at the heart of the metazoan NMD effector phase.

Nonsense Mediated mRNA Decay↗

PubMed1992

Detection of a nonsense mutation in the dystrophin gene by multiple SSCP.

A combination of multiplex PCR with the single strand conformation polymorphism (SSCP) technique was employed to screen for point mutations in the human dystrophin gene. Co-amplification of 11 exons from genomic DNA of Duchenne and Becker muscular dystrophy (DMD/BMD) patients with no deletion or duplication was performed and the samples subjected to multiple SSCP analysis. We report the case of a nonsense mutation in a Duchenne patient identified by this approach. The mutation introduces a termination codon within exon 8 of the dystrophin gene. It is predicted to cause a very premature translational termination accounting for the severe phenotype observed. The patient inherited this mutation from his mother. In addition the analysis revealed 5 polymorphisms useful for internal control.

Codon↗

PubMed1991

Mutants of Escherichia coli initiator tRNA that suppress amber codons in Saccharomyces cerevisiae and are aminoacylated with tyrosine by yeast extracts.

We recently described mutants of Escherichia coli initiator tRNA that suppress amber termination codons (UAG) in E. coli. These mutants have changes in the anticodon sequence (CAU----CUA) that allow them to read the amber codon and changes in the acceptor stem that allow them to bind to the ribosomal aminoacyl (A) site. We show here that a subset of these mutants suppress amber codons in Saccharomyces cerevisiae and that they are aminoacylated with tyrosine by yeast extracts. Analysis of a number of mutants as substrates for yeast tyrosyl-tRNA synthetase has led to identification of the C1.G72 base pair and the discriminator base A73, conserved in all eukaryotic cytoplasmic and archaebacterial tyrosine tRNAs, as being important for recognition. Our results suggest that the C1.G72 base pair and the discriminator base, in addition to the anticodon nucleotides previously identified [Bare, L.A. & Uhlenbeck, O.C. (1986) Biochemistry 25, 5825-5830] as important in yeast tyrosyl-tRNA synthetase recognition, may comprise the critical identity determinants in yeast tyrosine tRNA.

Amino Acyl-tRNA Synthetases↗

PubMed1993

The 3' codon context effect on UAG suppressor tRNA is different in Escherichia coli and human cells.

We have compared the effect of 3' context on the efficiency of nonsense suppressor tRNAs in Escherichia coli and human cells. Plasmids containing amber (UAG) termination codons were constructed in the vector pRSV beta gal by oligonucleotide insertion at an N-terminal location in a lacZ fusion. A family of identical vectors was prepared with either A, C, G or U as the first 3' base following the stop codon. These derivatives of pRSV beta gal were expressed in E. coli as stable plasmids, or transiently in human 293 cell tissue culture. Nonsense suppression was monitored using enzyme assays for beta-galactosidase. In E. coli the efficiency of a plasmid-borne bacterial tRNA(trp) UAG suppressor varied A > G > C = U. When the same lacZ reporter vectors were cotransfected with a human tRNA(ser) UAG suppressor plasmid into human cells, context effects of a different nature were detected. Double reciprocal analysis of dose-response experiments were used to show that the efficiency of suppression varied C > G > U = A. The discovery of different codon context effects on nonsense suppression in human cells suggest that the interaction between mammalian tRNAs or release factors and their target codons may have different characteristics from those in bacteria.

Base Sequence↗

PubMed1984

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone.

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.

Amino Acid Sequence↗

PubMed1993

Structure of the human lysyl oxidase gene.

Lysyl oxidase (EC 1.4.3.13), an extracellular copper enzyme, initiates the crosslinking of collagens and elastin by catalyzing oxidative deamination of the epsilon-amino group in certain lysine and hydroxylysine residues. We report here that the human lysyl oxidase gene is about 15 kb in size and consists of seven exons. Transcription is initiated at one major site and four minor sites, and the first exon consists of 273 bp of untranslated sequences (calculated to the major site) and 631 bp of translated sequences, which accounts for about half of all the translated sequences of the gene. The seventh exon, on the other hand, codes for only the last codon of amino acid 416 and for amino acid 417, which are followed by the translation termination codon and the 3' untranslated sequences. Exons 2-6 vary in size from 96 to 157 bp, and the introns from 331 bp to about 3.5 kb. The 5' flanking region contains a TATA-like sequence at -30 relative to the major transcription initiation site and a CCAAT motif at -109. The 5' flanking region and the downstream sequences present in the first exon and first intron contain altogether five possible binding sequences for Sp1, six for AP-2, one for AP-1, three for PEA3, three for MEP-1, and three CCCTCCC motifs, all of which may be involved in the regulation of the expression of the gene.

Amino Acid Sequence↗

PubMed1996

The gene organization, chromosome location, and expression of a 55-kDa matrix protein (PRELP) of human articular cartilage.

The gene corresponding to a 55-kDa matrix protein previously described in adult human articular cartilage was characterized by sequencing of genomic clones. The deduced protein sequence corresponds to the recently described matrix protein PRELP. The protein was encoded by messages of 1.7, 4.6, and 6.7 kb, whose relative abundance increased as their size decreased. The message heterogeneity appears to originate from variation in the length of the 3'-untranslated region, with the smallest message being contained within the reported sequence and the larger messages having extended 3'-untranslated regions. Two introns were identified within the genomic sequence encoding the smallest message. The first intron of about 6.7 kb resides 16 nucleotides prior to the translation initiation codon, and the second intron of about 2.6 kb resides 173 nucleotides prior to the translation termination codon. The gene, which encompasses at least 16 kb of genomic DNA, was shown to reside on chromosome 1q32. Primer extension techniques were used to establish that the coding sequence commences 199 bp downstream from the major transcription start site. Analysis of the DNA sequence upstream from the transcription start site reveals the presence of numerous potential transcription factor binding sites, but no CAAT or TATA box. At the message level, gene expression was at a high level in juvenile and adult cartilage, but not in the fetus or neonate. The presence of protein in the cartilage matrix was also much lower in the neonate than in the adult. In noncartilagenous tissues appreciable message levels were observed only in the adult lung.

Adult↗

PubMed1985

Functional analysis of lac repressor restart sites in translational initiation and reinitiation.

To define some of the features that influence ribosomal recognition of translational restart sites in the lac repressor mRNA, recombinant DNA methods have been used to construct lacI-Z fusions in which lacZ gene expression is dependent upon initiation or reinitiation within lacI mRNA sequences. Reinitiation efficiencies, as assessed by beta-galactosidase levels in strains bearing such plasmids, appear to be determined by at least three features of the RNA between the termination codon and reinitiation codon: the presence of competing out-of-frame AUG or GUG triplets, the distance between termination and reinitiation points, and the extent to which restart sequences remain accessible to ribosomes. While some of the restart sites are used with substantial efficiency for reinitiation, they do not function detectably as primary initiators if placed at the 5' end of the lacZ mRNA. This finding concurs with our observation that relative to the wild-type initiator region, which is recovered in quantitative yield from in vitro initiation reactions, ribosome protection of the four restart sites occurs at more than 100-fold lower efficiencies. In part, the lack of initiation activity is rationalized by the striking potential these sequences have for forming stable secondary structures that sequester elements essential for ribosome binding. However, the differential functioning of the restart sites in primary initiation versus reinitiation must also reflect real differences in the mechanisms operative in the two events.

Base Sequence↗

PubMed1998

A novel insertional mutation in loricrin in Vohwinkel's Keratoderma.

A mutation in the gene encoding loricrin has recently been reported in a subset of patients with Vohwinkel's Keratoderma manifesting an associated ichthyosiform dermatosis. We have studied a further kindred with this clinical phenotype. Microsatellite marker analysis was consistent with linkage to chromosome 1q21 and direct sequencing of loricrin identified a heterozygous mutation with an insertion of a T residue at codon 209. This mutation is predicted to produce a mutant protein with a frameshift of its terminal 107 amino acids and to be 22 amino acids longer than the wild-type protein due to a delayed termination codon. The only previously reported mutation is a G insertion producing a frameshift after codon 231. The novel mutation we report is likely to have a similar functional effect on cornified envelope formation, with disturbance of transglutaminase-mediated cross-linking of envelope components, and serves to confirm the predicted role of insertional mutations in Vohwinkel's Keratoderma associated with ichthyosis.

Amino Acid Sequence↗

PubMed2003

Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation.

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3' end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.

Codon↗

PubMed1989

Isolation and characterization of the gene coding for Escherichia coli arginyl-tRNA synthetase.

The gene coding for Escherichia coli arginyl-tRNA synthetase (argS) was isolated as a fragment of 2.4 kb after analysis and subcloning of recombinant plasmids from the Clarke and Carbon library. The clone bearing the gene overproduces arginyl-tRNA synthetase by a factor 100. This means that the enzyme represents more than 20% of the cellular total protein content. Sequencing revealed that the fragment contains a unique open reading frame of 1734 bp flanked at its 5' and 3' ends respectively by 247 bp and 397 bp. The length of the corresponding protein (577 aa) is well consistent with earlier Mr determination (about 70 kd). Primer extension analysis of the ArgRS mRNA by reverse transcriptase, located its 5' end respectively at 8 and 30 nucleotides downstream of a TATA and a TTGAC like element (CTGAC) and 60 nucleotides upstream of the unusual translation initiation codon GUG; nuclease S1 analysis located the 3'-end at 48 bp downstream of the translation termination codon. argS has a codon usage pattern typical for highly expressed E. coli genes. With the exception of the presence of a HVGH sequence similar to the HIGH consensus element, ArgRS has no relevant sequence homologies with other aminoacyl-tRNA synthetases.

Amino Acid Sequence↗

PubMed1996

Extensive RNA editing of U to C in addition to C to U substitution in the rbcL transcripts of hornwort chloroplasts and the origin of RNA editing in green plants.

We cloned and sequenced a portion of chloroplast DNA from the hornwort Anthoceros formosae. A nucleotide sequence of 7556 bp contained structures similar to those of ndhK, ndhC, trnV, trnM, atpE, atpB, rbcL, trnR and accD. The arrangement of these was the same as that of other chloroplast DNA. However, two nonsense codons were located within the putative coding region of rbcL, although they were used as putative termination codons of the genes. RNA was extensively edited in the transcripts of rbcL when cDNA sequences were analyzed. The unusual nonsense codons of TGA and TAA became CGA and CAA respectively. These are examples of U to C type RNA editing, which was never been found before in chloroplast mRNA. In general, 13 Cs of genomic DNA were found as Ts in the cDNA sequence and seven Ts were found as Cs. This is the first finding of RNA editing on the transcripts of rbcL and also in bryophytes. This event had been thought to arise in land plants after the split of bryophytes. The origin of RNA editing is discussed in relation to the landing of green plants.

Amino Acid Sequence↗

PubMed2006

Human alpha2-globin nonsense-mediated mRNA decay induced by a novel alpha-thalassaemia frameshift mutation at codon 22.

We describe a novel alpha-thalassaemia determinant in a 3-year-old girl presenting a mild microcytic and hypochromic anaemia, and normal haemoglobin A2 level. Molecular studies revealed heterozygosity for a novel microdeletion (-C) at codon 22 of the alpha2-globin gene. As the frameshift mutation generates a premature translation termination codon at position 48/49, we investigated the effect of the nonsense codon on the alpha2-globin gene expression. Although it does not affect RNA splicing, the premature nonsense codon induces accelerated mRNA degradation. To our knowledge, this is the first time the nonsense-mediated mRNA decay has been reported to occur in human alpha-globin mRNA.

Codon, Nonsense↗

PubMed1984

In vivo transcription of a human antithrombin III "minigene".

A 2.2-kilobase human antithrombin III (ATIII) "minigene" has been constructed which preserves many of the features of the 16-kilobase parental gene. These include approximately 300 base pairs of the 5'-flanking region containing the "TATA"-less ATIII promoter, the entire protein-coding sequence including the signal peptide, a single intervening sequence with intact donor and acceptor splice sites, and a polyadenylation signal. Unique PstI termini allow the minigene to be cloned in many commonly used plasmid vectors. The minigene has been reintroduced into heterologous cells by calcium phosphate precipitation. RNA is transcribed with heterogeneous 5'-ends and is processed by splicing either to the normal acceptor site or to an alternate site within the intervening sequence. The alternately spliced mRNA is also found in liver where it accounts for 20-40% of ATIII-specific transcripts. The alternate splicing event introduces a 42-nucleotide segment between codons -19 and -18 of the signal peptide. This segment contains an in-frame ochre termination codon such that the predicted protein product encoded by the alternately spliced mRNA would be only 19 amino acids long. The minigene is transcribed to high levels in mouse fibroblasts when present as an episome in a bovine papilloma virus-derived vector although no protein product is detected.

Amino Acid Sequence↗

PubMed1982

[Evolutionary changes in the genetic code, predictable on basis of the hypothesis of physical predetermination of the structure of codon bases].

According to the earlier proposed hypothesis on the structural correspondence between amino acids and doublets from the first codon bases (Sukhodolets 1980), the UGA triplet corresponds to tryptophan and the AGX triplets - to the termination codons. It is notably this sense of the UGA and AGA, AGG, respectively, that was reported for mitochondrial codes. Thereby, a proposal is indirectly confirmed that meanings of the UGA (nonsense) and AGA, AGG (arginine) in the normal cytoplasmic code is the result of evolutionary changes.

Amino Acids↗

PubMed1979

Five TGA "stop" codons occur within the translated sequence of the yeast mitochondrial gene for cytochrome c oxidase subunit II.

A mitochondrial mutation that genetically maps in the middle of the gene coding cytochrome c oxidase subunit II has been found to be a single-base-pair deletion. Three independently isolated spontaneous revertants of this mutant have different single-base-pair insertions within 15 nucleotides of the mutation. These findings clearly identify the location of the gene and suggest that the mutation causes a frame-shift. The sequence of about 900 base pairs surrounding the mutation has been determined and found to have several chain termination codons in every possible reading frame. The sequence can, however, be translated in one frame by assuming that the codon TGA does not cause chain termination in yeast mitochondira, as was recently suggested for the human organelle [Barrell, B. G., Bankier, A. T. & Drouin, J. (1979) Nature (London), in press]. If TGA codes for tryptophan residues, as is apparently the case in human mitochondria, a polypeptide can be read from the yeast mtDNA that is identical to bovine cytochrome oxidase subunit II at 37.8% of its residues. Furthermore, the DNA sequences of the frame-shift revertants discussed above predict relative isolectric point differences between the wild-type and various revertant forms of the polypeptide. The detection of these isolectric point differences by two-dimensional electrophoresis of subunit II from the various strains independently confirms the presumed reading frame of the gene. It is concluded that TGA is translated in yeast mitochondria, most probably as tryptophan.

Base Sequence↗