Driving through stop signs: predicting stop codon reassignment improves functional annotation of bacteriophages
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PROVIDER: S-EPMC11210395 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:The majority of bacteriophage diversity remains uncharacterised, and new intriguing mechanisms of their biology are being continually described. Members of some phage lineages, such as the Crassvirales, repurpose stop codons to encode an amino acid by using alternate genetic codes. Here, we investigated the prevalence of stop codon reassignment in phage genomes and subsequent impacts on functional annotation. We predicted 76 genomes within INPHARED and 712 vOTUs from the Unified Human Gut Virome catalogue (UHGV) that repurpose a stop codon to encode an amino acid. We re-annotated these sequences with modified versions of Pharokka and Prokka, called Pharokka-gv and Prokka-gv, to automatically predict stop codon reassignment prior to annotation. Both tools significantly improved the quality of annotations, with Pharokka-gv performing best. For sequences predicted to repurpose TAG to glutamine (translation table 15), Pharokka-gv increased the median gene length (median of per genome medians) from 287 to 481 bp for UHGV sequences (67.8% increase) and from 318 to 550 bp for INPHARED sequences (72.9% increase). The re-annotation increased mean coding density from 66.8% to 90.0%, and from 69.0% to 89.8% for UHGV and INPHARED sequences. Furthermore, the proportion of genes that could be assigned functional annotation increased, including an increase in the number of major capsid proteins that could be identified. We propose that automatic prediction of stop codon reassignment before annotation is beneficial to downstream viral genomic and metagenomic analyses.
| S-EPMC10769273 | biostudies-literature
Project description:BackgroundEfficient gene expression involves a trade-off between (i) premature termination of protein synthesis; and (ii) readthrough, where the ribosome fails to dissociate at the terminal stop. Sense codons that are similar in sequence to stop codons are more susceptible to nonsense mutation, and are also likely to be more susceptible to transcriptional or translational errors causing premature termination. We therefore expect this trade-off to be influenced by the number of stop codons in the genetic code. Although genetic codes are highly constrained, stop codon number appears to be their most volatile feature.ResultsIn the human genome, codons readily mutable to stops are underrepresented in coding sequences. We construct a simple mathematical model based on the relative likelihoods of premature termination and readthrough. When readthrough occurs, the resultant protein has a tail of amino acid residues incorrectly added to the C-terminus. Our results depend strongly on the number of stop codons in the genetic code. When the code has more stop codons, premature termination is relatively more likely, particularly for longer genes. When the code has fewer stop codons, the length of the tail added by readthrough will, on average, be longer, and thus more deleterious. Comparative analysis of taxa with a range of stop codon numbers suggests that genomes whose code includes more stop codons have shorter coding sequences.ConclusionsWe suggest that the differing trade-offs presented by alternative genetic codes may result in differences in genome structure. More speculatively, multiple stop codons may mitigate readthrough, counteracting the disadvantage of a higher rate of nonsense mutation. This could help explain the puzzling overrepresentation of stop codons in the canonical genetic code and most variants.
| S-EPMC3161013 | biostudies-literature
Project description:Recoding of stop codons as amino acid-specifying codons is a co-translational event that enables C-terminal extension of a protein. Synthesis of selenoproteins requires recoding of internal UGA stop codons to the 21st non-standard amino acid selenocysteine (Sec) and plays a vital role in human health and disease. Separately, canonical stop codons can be recoded to specify standard amino acids in a process known as stop codon readthrough (SCR), producing extended protein isoforms with potential novel functions. Conventional computational tools cannot distinguish between the dual functionality of stop codons as stop signals and sense codons, resulting in misannotation of selenoprotein gene products and failure to predict SCR. Manual curation is therefore required to correctly represent recoded gene products and their functions. Our goal was to provide accurately curated and annotated datasets of selenoprotein and SCR transcript and protein records to serve as annotation standards and to promote basic and biomedical research. Gene annotations were curated in nine vertebrate model organisms and integrated into NCBI's Reference Sequence (RefSeq) dataset, resulting in 247 selenoprotein genes encoding 322 selenoproteins, and 93 genes exhibiting SCR encoding 94 SCR isoforms.
| S-EPMC6344875 | biostudies-literature
Project description:Bacteriophages (phages) are obligate parasites that use host bacterial translation machinery to produce viral proteins. However, some phages have alternative genetic codes with reassigned stop codons that are predicted to be incompatible with bacterial translation systems. We analysed 9,422 phage genomes and found that stop-codon recoding has evolved in diverse clades of phages that infect bacteria present in both human and animal gut microbiota. Recoded stop codons are particularly over-represented in phage structural and lysis genes. We propose that recoded stop codons might function to prevent premature production of late-stage proteins. Stop-codon recoding has evolved several times in closely related lineages, which suggests that adaptive recoding can occur over very short evolutionary timescales.
| S-EPMC9197471 | biostudies-literature
Project description:In the mitochondrial genome of the hemichordate Balanoglossus carnosus, the codon AAA, which is assigned to lysine in most metazoans but to asparagine in echinoderms, is absent. Furthermore, the lysine tRNA gene carries an anticodon substitution that renders its gene product unable to decode AAA codons, whereas the asparagine tRNA gene has not changed to encode a tRNA with the ability to recognize AAA codons. Thus, the hemichordate mitochondrial genome can be regarded as an intermediate in the process of reassignment of mitochondrial AAA codons, where most metazoans represent the ancestral situation and the echinoderms the derived situation. This lends support to the codon capture hypothesis. We also show that the reassignment of the AAA codon is associated with a reduction in the relative abundance of lysine residues in mitochondrial proteins.
| S-EPMC19900 | biostudies-literature
Project description:Over the last decade, the ability to genetically encode unnatural amino acids (UAAs) has evolved rapidly. The programmed incorporation of UAAs into recombinant proteins relies on the reassignment or suppression of canonical codons with an amino-acyl tRNA synthetase/tRNA (aaRS/tRNA) pair, selective for the UAA of choice. In order to achieve selective incorporation, the aaRS should be selective for the designed tRNA and UAA over the endogenous amino acids and tRNAs. Enhanced selectivity has been achieved by transferring an aaRS/tRNA pair from another kingdom to the organism of interest, and subsequent aaRS evolution to acquire enhanced selectivity for the desired UAA. Today, over 150 non-canonical amino acids have been incorporated using such methods. This enables the introduction of a large variety of structures into proteins, in organisms ranging from prokaryote, yeast and mammalian cells lines to whole animals, enabling the study of protein function at a level that could not previously be achieved. While most research to date has focused on the suppression of 'non-sense' codons, recent developments are beginning to open up the possibility of quadruplet codon decoding and the more selective reassignment of sense codons, offering a potentially powerful tool for incorporating multiple amino acids. Here, we aim to provide a focused review of methods for UAA incorporation with an emphasis in particular on the different tRNA synthetase/tRNA pairs exploited or developed, focusing upon the different UAA structures that have been incorporated and the logic behind the design and future creation of such systems. Our hope is that this will help rationalize the design of systems for incorporation of unexplored unnatural amino acids, as well as novel applications for those already known.
| S-EPMC5424465 | biostudies-literature
Project description:It is widely hypothesized that removing cellular transfer RNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.
| S-EPMC7611380 | biostudies-literature
Project description:Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNAOpt We suspected a modification of the tRNAOptAUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNAOptAUG is converted to inosine. We identified tRNAOptAUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms.
| S-EPMC5137457 | biostudies-literature
Project description:INTRODUCTION: Dectin-1, a pattern recognition receptor expressed by the innate immune system, is known to be a major receptor inducing Th17-type adaptive immune responses that have been demonstrated to mediate autoimmunity. In this study, dectin-1 mRNA and protein expression, as well as the recently characterized DECTIN-1 Y238X early stop codon polymorphism, were studied in relation to rheumatoid arthritis (RA) susceptibility and severity. METHODS: Dectin-1 mRNA expression was measured in synovial tissue specimens of RA, osteoarthritis (OA), and nonrheumatic patients. Dectin-1 protein expression and localization were assessed in RA synovial tissue specimens. Macrophages from individuals with different DECTIN-1 genotypes were examined for differences in cytokine responses on dectin-1 stimulation. Furthermore, clinical parameters of inflammation and bone destruction of 262 RA patients were correlated with the presence of the DECTIN-1 Y238X polymorphism. RESULTS: Evaluation of dectin-1 mRNA expression in synovial tissue biopsies revealed an increased expression in RA specimens, compared with biopsies from OA and nonrheumatic patients. Accordingly, dectin-1 protein expression in RA synovial tissue biopsies was moderate to high, especially on macrophage-like cells. Cytokine production capacity of macrophages bearing the DECTIN-1 Y238X polymorphism was demonstrated to be impaired on dectin-1 stimulation. However, the presence of the DECTIN-1 Y238X polymorphism was not associated with RA susceptibility or disease severity. CONCLUSIONS: Although expression of dectin-1 was high in synovial tissue of RA patients, and reduced cytokine production was observed in macrophages of individuals bearing the DECTIN-1 Y238X polymorphism, loss of one functional allele of DECTIN-1 is not associated with either susceptibility to or severity of RA.
| S-EPMC2875660 | biostudies-literature
Project description:Nonsense suppression therapy encompasses approaches aimed at suppressing translation termination at in-frame premature termination codons (PTCs, also known as nonsense mutations) to restore deficient protein function. In this review, we examine the current status of PTC suppression as a therapy for genetic diseases caused by nonsense mutations. We discuss what is currently known about the mechanism of PTC suppression as well as therapeutic approaches under development to suppress PTCs. The approaches considered include readthrough drugs, suppressor tRNAs, PTC pseudouridylation, and inhibition of nonsense-mediated mRNA decay. We also discuss the barriers that currently limit the clinical application of nonsense suppression therapy and suggest how some of these difficulties may be overcome. Finally, we consider how PTC suppression may play a role in the clinical treatment of genetic diseases caused by nonsense mutations.
| S-EPMC5304456 | biostudies-literature