Canonical Stop Codon Research Articles

Haematococcus lacustris: the makings of a giant-sized chloroplast genome.

Recent work on the chlamydomonadalean green alga Haematococcus lacustris uncovered the largest plastid genome on record: a whopping 1.35 Mb with >90 % non-coding DNA. A 500-word description of this genome was published in the journal Genome Announcements. But such a short report for such a large genome leaves many unanswered questions. For instance, the H. lacustris plastome was found to encode only 12 tRNAs, less than half that of a typical plastome, it appears to have a non-standard genetic code, and is one of only a few known plastid DNAs (ptDNAs), out of thousands of available sequences, not biased in adenine and thymine. Here, I take a closer look at the H. lacustris plastome, comparing its size, content and architecture to other large organelle DNAs, including those from close relatives in the Chlamydomonadales. I show that the H. lacustris plastid coding repertoire is not as unusual as initially thought, representing a standard set of rRNAs, tRNAs and protein-coding genes, where the canonical stop codon UGA appears to sometimes signify tryptophan. The intergenic spacers are dense with repeats, and it is within these regions where potential answers to the source of such extreme genomic expansion lie. By comparing ptDNA sequences of two closely related strains of H. lacustris, I argue that the mutation rate of the non-coding DNA is high and contributing to plastome inflation. Finally, by exploring publicly available RNA-sequencing data, I find that most of the intergenic ptDNA is transcriptionally active.

Phylogenetically Conserved Sequences Around Myelin P0 Stop Codon are Essential for Translational Readthrough to Produce L-MPZ.

Myelin protein zero (P0, MPZ) is the main cell adhesion molecule in peripheral myelin, the sequence of which is evolutionarily highly conserved. Large myelin protein zero (L-MPZ) is a novel translational readthrough molecule in mammals in a physiological status and is encoded by the P0 mRNA with an extra domain. The sequence similarities in the L-MPZ-specific region are found in humans and frogs but not in fish P0 cDNA. Actual synthesis of L-MPZ has been detected in rat and mouse sciatic nerve but not yet evaluated in frogs and humans. The production mechanism and physiological functions of L-MPZ remain unknown. Additionally, the sequence context around the canonical stop codon is significant for readthrough in viruses and yeast, but the correlation between the sequence around P0 stop codon and L-MPZ synthesis is unclear. Here, we focused on the phylogenetic pathways in L-MPZ synthesis. We have shown that L-MPZ is widely produced from frogs to humans using western blotting against L-MPZ. Mutation analysis of the sequence around the stop codon for L-MPZ synthesis using a mammalian in vitro transcription/translation system revealed that the evolutionarily conserved sequence around P0 stop codon is susceptible to readthrough and is similar to the consensus motif in viruses and yeast UAG stop codon type molecules. Our results demonstrate that the phylogenetically conserved sequence around the canonical P0 stop codon is essential for L-MPZ synthesis, suggesting that phylogenetic emergence of L-MPZ in amphibians may be related to particular distribution and/or function in the PNS myelin.

Influence of negamycin-derived stop codon readthrough agents on physiological readthrough event in vivo

Background: Suppression of nonsense mutations using stop codon readthrough drugs is one of the promising treatments for various genetic disorders, including Duchenne muscular dystrophy. However, recent studies indicate that some functional proteins are produced by canonical stop codon readthrough in physiological conditions of human and other animals. Large myelin protein zero (L-MPZ) is one of these proteins. It contains extra 63 amino acids at C-terminus of myelin protein zero (P0), which is a major myelin protein in the peripheral nervous system. Adhesion activity of L-MPZ is less than that of P0, suggesting that the ratio of these two proteins in myelin is important for normal myelin structure and function.

Comparative mitochondrial genome analysis of Daphnis nerii and other lepidopteran insects reveals conserved mitochondrial genome organization and phylogenetic relationships.

In the present study, the complete sequence of the mitochondrial genome (mitogenome) of Daphnis nerii (Lepidoptera: Sphingidae) is described. The mitogenome (15,247 bp) of D.nerii encodes13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and an adenine (A) + thymine (T)-rich region. Its gene complement and order is similar to that of other sequenced lepidopterans. The 12 PCGs initiated by ATN codons except for cytochrome c oxidase subunit 1 (cox1) gene that is seemingly initiated by the CGA codon as documented in other insect mitogenomes. Four of the 13 PCGs have the incomplete termination codon T, while the remainder terminated with the canonical stop codon. This mitogenome has six major intergenic spacers, with the exception of A+T-rich region, spanning at least 10 bp. The A+T-rich region is 351 bp long, and contains some conserved regions, including ‘ATAGA’ motif followed by a 17 bp poly-T stretch, a microsatellite-like element (AT)9 and also a poly-A element. Phylogenetic analyses based on 13 PCGs using maximum likelihood (ML) and Bayesian inference (BI) revealed that D. nerii resides in the Sphingidae family.

Therapeutic Suppression of Nonsense Mutation: An Emerging Target in Multiple Diseases and Thrombotic Disorders.

Nonsense mutations contribute to approximately 10-30% of the total human inherited diseases via disruption of protein translation. If any of the three termination codons (UGA, UAG and UAA) emerges prematurely [known as premature termination codon (PTC)] before the natural canonical stop codon, truncated nonfunctional proteins or proteins with deleterious loss or gain-of-function activities are synthesized, followed by the development of nonsense mutation-mediated diseases. In the past decade, PTC-associated diseases captured much attention in biomedical research, especially as molecular therapeutic targets via nonsense suppression (i.e. translational readthrough) regimens. In this review, we highlighted different treatment strategies of PTC targeting readthrough therapeutics including the use of aminoglycosides, ataluren (formerly known as PTC124), suppressor tRNAs, nonsense-mediated mRNA decay, pseudouridylation and CRISPR/Cas9 system to treat PTC-mediated diseases. In addition, as thrombotic disorders are a group of disease with major burdens worldwide, 19 potential genes containing a total of 705 PTCs that cause 21 thrombotic disorders have been listed based on the data reanalysis from the 'GeneCards® - Human Gene Database' and 'Human Gene Mutation Database' (HGMD®). These PTC-containing genes can be potential targets amenable for different readthrough therapeutic strategies in the future.

Mitochondrial genome of Abraxas suspecta (Lepidoptera: Geometridae)and comparative analysis with other Lepidopterans.

In this study, a complete mitochondrial genome (mitogenome) sequence of Abraxas suspecta (Lepidoptera: Geometridae) is isolated and characterized. The complete DNA is 15,547 bp length and contains 2 ribosomal RNA genes, 23 putative transfer RNA (tRNA) genes including an extra tRNAAsn (AUU), 13 protein-coding genes and an adenine (A) + thymine (T)-rich region. The nucleotide composition and gene organization are identical to those of other lepidopteran, except for the presence of an extra copy of trnN (AUU). Of the 38 genes, twenty-five genes (9 PCGs and 16 tRNAs) are encoded by heavy strand (H-strand), while thirteen are encoded by light strand (L-strand). Among the 13 PCGs, 12 PCGs employ ATN as initiation codon, while cytochrome c oxidase subunit 1 (cox1) utilizes CGA as initiation codon. Four of the 13 PCGs have the incomplete termination codon T, while the remainder terminated with the canonical stop codon. All tRNA genes are folded into the typical clover-leaf structure of mitochondrial tRNAs, except for the tRNASer (AGN) gene, in which the DHU arm fails to form a stable stem-loop structure. The A+T-rich region is 532 bp long, and contains some conserved regions, including 'ATAGA' motif followed by a 17bp poly-T stretch, a microsatellite-like element (AT)8(AAT)3 and also a poly-A element. A short Phylogenetic analysis based on 13 PCGs using maximum likelihood (ML) and Bayesian inference (BI) revealed that A. suspecta resides in the Geometridae family. We present the method and approach to use moths as model organisms for further genetic and evolutionary biology studies.

First complete mitochondrial genomes of molossid bats (Chiroptera: Molossidae)

Bats represent around one-fourth of the world’s mammals and their taxonomy is still controversial. Molossids are one of the most diverse bat families with a wide knowledge gap. In this study, we report the first complete mitochondrial genomes of three molossid bats: the European free-tailed bat Tadarida teniotis, the La Touche’s free-tailed bat Tadarida latouchei, and the Wrinkle-lipped free-tailed bat Chaerephon plicatus. The mitogenomes are 16,869 and 16,784 bp long for T. teniotis and T. latouchei, respectively, while in C. plicatus it is at least 16,216 bp although the control region was not fully recovered due to its higher divergence from T. teniotis. The genomes show conserved synteny with other mammalian mitogenomes, containing 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 control region (d-loop). All protein-coding genes start with the ATG start codon, except for ND2, ND3, and ND5 which begin with ATA or ATT. Eleven protein-coding genes terminated in a canonical stop codon, TAA or TAG, two contain incomplete stop codons, T or TA. Cytochrome b terminates in the mitochondria-specific stop codon AGA. These mitogenomes provide a valuable resource for future studies of Molossidae and other bat and mammal species.

Crystal structures of the human elongation factor eEFSec suggest a non-canonical mechanism for selenocysteine incorporation.

Selenocysteine is the only proteinogenic amino acid encoded by a recoded in-frame UGA codon that does not operate as the canonical opal stop codon. A specialized translation elongation factor, eEFSec in eukaryotes and SelB in prokaryotes, promotes selenocysteine incorporation into selenoproteins by a still poorly understood mechanism. Our structural and biochemical results reveal that four domains of human eEFSec fold into a chalice-like structure that has similar binding affinities for GDP, GTP and other guanine nucleotides. Surprisingly, unlike in eEF1A and EF-Tu, the guanine nucleotide exchange does not cause a major conformational change in domain 1 of eEFSec, but instead induces a swing of domain 4. We propose that eEFSec employs a non-canonical mechanism involving the distinct C-terminal domain 4 for the release of the selenocysteinyl-tRNA during decoding on the ribosome.

Structural Basis for Translation Termination on a Pseudouridylated Stop Codon

Pseudouridylation of messenger RNA emerges as an abundant modification involved in gene expression regulation. Pseudouridylation of stop codons in eukaryotic and bacterial cells results in stop-codon read through. The structural mechanism of this phenomenon is not known. Here we present a 3.1-Å crystal structure of Escherichia coli release factor 1 (RF1) bound to the 70S ribosome in response to the ΨAA codon. The structure reveals that recognition of a modified stop codon does not differ from that of a canonical stop codon. Our in vitro biochemical results support this finding by yielding nearly identical rates for peptide release from E. coli ribosomes programmed with pseudouridylated and canonical stop codons. The crystal structure also brings insight into E. coli RF1-specific interactions and suggests involvement of L27 in bacterial translation termination. Our results are consistent with a mechanism in which read through of a pseudouridylated stop codon in bacteria results from increased decoding by near-cognate tRNAs (miscoding) rather than from decreased efficiency of termination.

The complete mitochondrial genome of the Greater Mouse-Eared bat, Myotis myotis (Chiroptera: Vespertilionidae)

In this study, we report the complete mitochondrial genome of the Greater Mouse-Eared Bat, Myotis myotis. The mitogenome is 17 213 bp with base composition A (34.2%), G (13%), C (22.4%), and T (30.5%). The genome shows conserved synteny with other mammalian mitogenomes, containing 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 control region (D-Loop). The majority of the genes are encoded on the H-Strand, except for ND6 and eight tRNAs. All protein-coding genes start with the ATG start codon, except for ND2, ND3, and ND5 which begin with ATT or ATA. Seven protein-coding genes terminated in a canonical stop codon, TAA or TAG, five contain incomplete stop codons, T or TA. Cytochrome b terminates in the mitochondria specific stop codon AGA. This mitogenome provides a valuable resource for future studies of M. myotis and other bat and mammal species.

Antiangiogenic VEGF-Ax: A New Participant in Tumor Angiogenesis.

The transcript of the angiogenic factor vascular endothelial growth factor A (VEGF-A) is subject to a multitude of stimulus-dependent, posttranscriptional regulatory events, consistent with its unusually long 3' untranslated region. We have recently reported translational readthrough of VEGFA mRNA whereby translating ribosomes traverse the canonical stop codon to a conserved, downstream stop codon, generating VEGF-Ax ("x" for extended), a novel, extended isoform with an additional 22 amino acids appended at the C-terminus. This event is the first vertebrate example of protein-regulated, programmed translational readthrough that generates a protein with a known function. Remarkably, VEGF-Ax exhibits potent antiangiogenic activity, both in vitro and in vivo, thus raising profound clinical implications, particularly with respect to cancer treatment. In this review, we discuss the potential of VEGF-Ax as a therapeutic agent and drug target, as well as its possible role in the failure of, or resistance to, conventional anti-VEGF therapies in many types of cancers.

The complete mitochondrial genomes of two orb-weaving spider Cyrtarachne nagasakiensis (Strand, 1918) and Hypsosinga pygmaea (Sundevall, 1831) (Araneae: Araneidae)

The complete mitogenomes of Cyrtarachne nagasakiensis and Hypsosinga pygmaea are determined to be 14,402 and 14,193 bp in length, with the A + T content of 75.7% and 76.1%, respectively. All protein-coding genes in both spiders start with the initiation codons (ATT, ATA, TTA or TTG) that are usually used in Araneae mitogenomes, and terminate with canonical stop codon TAA or TAG except for ND5 in both spiders and ND4L in H. pygmaea, which end with the incomplete codon TA or T instead. Most of the tRNAs lack the potential to form the typically cloverleaf-shaped secondary structures and none of tandem repeats are present in the control regions of both spider mitogenomes. The phylogenetic analysis based on the Bayesian inference dataset shows a good resolution of relationship of C. nagasakiensis and H. pygmaea in Araneidae.

The complete mitochondrial genome of Rondotia menciana (Lepidoptera: Bombycidae).

The mulberry white caterpillar, Rondotia menciana Moore (Lepidoptera: Bombycidae) is a species with closest relationship with Bombyx mori and Bombyx mandarina, and the genetic information of R. menciana is important for understanding the diversity of the Bombycidae. In this study, the mitochondrial genome (mitogenome) of R. menciana was amplified by polymerase chain reaction and sequenced. The mitogenome of R. menciana was determined to be 15,301 bp, including 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes, and an AT-rich region. The A+T content (78.87%) was lower than that observed for other Bombycidae insects. All PCGs were initiated by ATN codons and terminated with the canonical stop codons, except for coxII, which was terminated by a single T. All the tRNA genes displayed a typical clover-leaf structure of mitochondrial tRNA. The length of AT-rich region (360 bp) of R. menciana mitogenome is shorter than that of other Bombycidae species. Phylogenetic analysis showed that the R. menciana was clustered on one branch with B. mori and B. mandarina from Bombycidae.

The complete mitochondrial genome of the orb-weaving spider Neoscona theisi (Walckenaer) (Araneae: Araneidae)

The complete mitochondrial genome of an orb-weaving spider Neoscona theisi (Walckenaer) is determined in this article. It is a typical circular duplex DNA molecule with a length of 14,156 bp, which encodes the same 37 genes as all metazoan mitogenomes and an A + T-rich region (D-loop). The overall A + T content is 75.2% (A: 35.5%; T: 39.8%; G: 15.6%; C: 9.3%). All of the protein-coding genes were initiated by ATN, with the exceptions in three genes. COI has a TTA start codon, COII, COIII and ND6 use TTG as initiation codon. Three genes (ND3, COII and ND5) end with incomplete stop codons (T or TA), while all other genes terminated with canonical stop codons (TAA or TAG). Among 22 transfer RNAs genes, six of them (tRNAAla, tRNASer(AGN), tRNASer(UCN), tRNAGly, tRNAArg and tRNAHis) lack the potential to form the cloverleaf-shaped secondary structure. The A + T-rich region is 559 bp with an A + T content of 79.6%.

Complete mitochondrial genome sequence of Heliconius melpomene rosina (Insecta: Lepidoptera: Nymphalidae)

The Postman Butterfly (Heliconius melpomene) is one of the heliconiine butterflies found in the Central and South America. In this study, the complete mitochondrial genome sequence of its subspecies, Heliconius melpomene rosina, is determined for the first time. Results show that this circular genome is 15,327 bp in length, and consists of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and a putative control region (D-loop). The genome organization, nucleotide composition and codon usage are identical to those reported from other butterfly mitochondrial genomes. The whole nucleotide composition is 39.33% of A, 42.33% of T, 10.88% of C and 7.46% of G, with a relatively lower level of G and an extremely higher AT content of 81.66%. Most of the PCGs initiate with the ATN start codons except for ND1 and COX1 genes, which separately start with TTG and CGA. Totally 13 PCGs terminate with the standard canonical stop codons of TAA/TAG/TGA or an incomplete stop codon of T– –. The mitochondrial genome sequence reported here would be potentially useful for the phylogenetic studies in butterflies.

The complete mitochondrial genome of fungus-growing termite, Macrotermes natalensis (Isoptera: Macrotermitinae)

In this study, the complete mitochondrial genome of a fungus-growing termite, Macrotermes natalensis, with the total length of 16,325 bp, is reported for the first time. This mtgenome harbors 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). The total base composition is 44.1% of A, 21.5% of T, 11.5% of G, and 22.9% of C, so the percentage of A and T (65.6%) is much higher than that of G and C. Most of the genes are distributed on H-strand, except for four subunit genes (ND1, ND4, ND4L, ND5) and eight tRNA genes. All protein-coding genes start with an ATN codon, and terminate with the canonical stop codon (TAA/TAG) or a single T (T– –). The complete mitochondrial genome sequence reported here adds a new genetic resource for the genus Macrotermes and might be useful for phylogenetic and systematic analyses within the genus Macrotermes.

Programmed Translational Readthrough Generates Antiangiogenic VEGF-Ax

Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here, we show that vascular endothelial growth factor A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino-acid C terminus extension. A cis-acting element in the VEGFA 3' UTR serves a dual function, not only encoding the appended peptide but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits antiangiogenic activity in contrast to the proangiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system.

The pyrrolysine translational machinery as a genetic-code expansion tool

The discovery of pyrrolysine not only expanded the set of the known proteinogenic amino acids but also revealed unusual features of its encoding mechanism. The engagement of a canonical stop codon and a unique aminoacyl-tRNA synthetase-tRNA pair that can be used to accommodate a broad range of unnatural amino acids while maintaining strict orthogonality in a variety of prokaryotic and eukaryotic expression systems has proven an invaluable combination. Within a few years since its properties were elucidated, the pyrrolysine translational machinery has become a popular choice for the synthesis of recombinant proteins bearing a wide variety of otherwise hard-to-introduce functional groups. It is also central to the development of new synthetic strategies that rely on stop-codon suppression.

A unique genetic code change in the mitochondrial genome of the parasitic nematode Radopholus similis

BackgroundMitochondria (mt) contain their own autonomously replicating DNA, constituted as a small circular genome encoding essential subunits of the respiratory chain. Mt DNA is characterized by a genetic code which differs from the standard one. Interestingly, the mt genome of nematodes share some peculiar features, such as small transfer RNAs, truncated ribosomal RNAs and - in the class of Chromadorean nematodes - unidirectional transcription.FindingsWe present the complete mt genomic sequence (16,791 bp) of the plant-parasitic nematode Radopholus similis (class Chromadorea). Although it has a gene content similar to most other nematodes, many idiosyncrasies characterize the extremely AT-rich mt genome of R. similis (85.4% AT). The secondary structure of the large (16S) rRNA is further reduced, the gene order is unique, the large non-coding region contains two large repeats, and most interestingly, the UAA codon is reassigned from translation termination to tyrosine. In addition, 7 out of 12 protein-coding genes lack a canonical stop codon and analysis of transcriptional data showed the absence of polyadenylation. Northern blot analysis confirmed that only one strand is transcribed and processed. Furthermore, using nucleotide content bias methods, regions for the origin of replication are suggested.ConclusionThe extraordinary mt genome of R. similis with its unique genetic code appears to contain exceptional features correlated to DNA decoding. Therefore the genome may provide an incentive to further elucidate these barely understood processes in nematodes. This comprehension may eventually lead to parasitic nematode-specific control targets as healthy mitochondria are imperative for organism survival. In addition, the presented genome is an interesting exceptional event in genetic code evolution.

The Highly Reduced and Fragmented Mitochondrial Genome of the Early-branching Dinoflagellate Oxyrrhis marina Shares Characteristics with both Apicomplexan and Dinoflagellate Mitochondrial Genomes

The mitochondrial genome and the expression of the genes within it have evolved to be highly unusual in several lineages. Within alveolates, apicomplexans and dinoflagellates share the most reduced mitochondrial gene content on record, but differ from one another in organisation and function. To clarify how these characteristics originated, we examined mitochondrial genome form and expression in a key lineage that arose close to the divergence of apicomplexans and dinoflagellates, Oxyrrhis marina. We show that Oxyrrhis is a basal member of the dinoflagellate lineage whose mitochondrial genome has some unique characteristics while sharing others with apicomplexans or dinoflagellates. Specifically, Oxyrrhis has the smallest gene complement known, with several rRNA fragments and only two protein coding genes, cox1 and a cob-cox3 fusion. The genome appears to be highly fragmented, like that of dinoflagellates, but genes are frequently arranged as tandem copies, reminiscent of the repeating nature of the Plasmodium genome. In dinoflagellates and Oxyrrhis, genes are found in many arrangements, but the Oxyrrhis genome appears to be more structured, since neighbouring genes or gene fragments are invariably the same: cox1 and the cob-cox3 fusion were never found on the same genomic fragment. Analysing hundreds of cDNAs for both genes and circularized mRNAs from cob-cox3 showed that neither uses canonical start or stop codons, although a UAA terminator is created in the cob-cox3 fusion mRNA by post-transcriptional oligoadenylation. mRNAs from both genes also use a novel 5′ oligo(U) cap. Extensive RNA editing is characteristic of dinoflagellates, but we find no editing in Oxyrrhis. Overall, the combination of characteristics found in the Oxyrrhis genome allows us to plot the sequence of many events that led to the extreme organisation of apicomplexan and dinoflalgellate mitochondrial genomes.