Codon Identity Research Articles

Non-random arrangement of synonymous codons in archaea coding sequences

Non-random arrangement of synonymous codons in coding sequences has been recently reported in eukaryotic and bacterial genomes, but the case in archaeal genomes is largely undetermined. Here, we systematically investigated 122 archaeal genomes for their synonymous codon co-occurrence patterns. We found that in most archaeal coding sequences, the order of synonymous codons is not arranged randomly, but rather some successive codon pairs appear significantly more often than expected. Importantly, such codon pairing bias (CPB) pattern in archaea does not seem to completely follow the co-tRNA codon pairing (CCP) rule previously reported for eukaryotes, but largely obeys an identical codon pairing (ICP) rule. Further, synonymous codon permutation test demonstrated that in many archaeal genomes, random mutation alone is unable to cause the observed high level of ICP bias, which strongly indicates that selection force has been involved to shape synonymous codon orders, potentially meeting a global requirement to optimize translation rate.

On the Limitations of Using Ribosomal Genes as References for the Study of Codon Usage: A Rebuttal

In a recent paper published in PLOS ONE, Wang et al. challenge our finding that the identity of optimal codons in different genomes follows a set of clear rules. Here we provide a rebuttal of their paper and demonstrate that the results of our original PLOS Genetics paper stand. This provides us with an opportunity to bring up an aspect of how codon usage has been studied that should be of general interest. The Wang et al. study, as well as many other studies, used ribosomal genes as a reference set for the study of patterns of codon usage. We discuss here the assumptions that are made in order to justify using ribosomal genes to study codon bias, suggest that this practice can at times be problematic, and discuss its limitations.

Selective pressure dominates the synonymous codon usage in parvoviridae

Parvoviridae is a family of small non-enveloped viruses and divided into two subfamilies. The family members infect a wide range of organisms from insects to humans and some of the members (e.g., nonpathogenic adeno-associated viruses) are effective gene therapy delivery vectors. We detailed the synonymous codon usage pattern of Parvoviridae family from the available 58 sequenced genomes through multivariate statistical methods. Our results revealed that nine viruses showed some degree of strong codon bias, and the others possessed a general weak trend of codon bias. ENc-plot and neutrality plot results showed that selective pressure dominated over mutation in shapes coding sequence's composition. The overall GC content and GC content at the third synonymous codon position were the principal determinants behind the variations within the codon usage patterns, as they both significantly correlated with the first axis of correspondence analysis. In addition, gene length had no direct influence on the codon usage pattern. Densovirinae subfamily and Parvovirinae subfamily possessed nine identical preferred codons, though most of the two subfamilies codon usage frequencies were significantly different. The result of cluster analysis based on synonymous codon usage was discordant with that of taxonomic classification. Adeno-associated viruses formed a separated clade far from other Parvoviridae members in the dendrogram. Thus, we concluded that natural selection rather than mutation pressure accounts for the main factor that affects the codon bias in Parvoviridae family.

Aminoglycoside-induced readthrough of stop codons in vivo elicits presentation of novel immunogenic MHC class I epitopes . (106.39)

Abstract Aminoglycoside antibiotics are known to induce stop codon readthrough in eukaryotic cells, and thus hold potential for the treatment of genetic diseases caused by nonsense mutations. A potential consequence of aminoglycoside treatment is the translation of 3’ noncoding regions and generation of major histocompatibiltiy complex (MHC)-restricted neoepitopes, a potential cause of autoimmune reactivity. Here we explored the impact of aminoglycoside-induced readthough on the production of MHC class I binding peptides. Firstly, we developed a panel of recombinant vaccinia viruses in which expression of a MHC class I binding peptide depends upon stop codon readthrough. Both in vitro and in vivo studies indicated that gentamicin induces activating levels of epitope with the magnitude of the response strongly influenced by identity of the stop codon and the surrounding context in agreement with a previously determined hierarchy. Secondly, we treated uninfected cells with gentamicin to assess the level of aminoglycoside-induced readthrough on native stop codons. HPLC analysis revealed a small number of differences in the peak profiles. Further analysis will be done with mass spectrometry to determine the identity of these epitopes, and where they are located in the mouse genome. The data from these and future studies can be used determine the extent to which stop codon readthrough occurs, and the implications for eliciting autoimmunity.

Statistical Analysis of Readthrough Levels for Nonsense Mutations in Mammalian Cells Reveals a Major Determinant of Response to Gentamicin

The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the −1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable.

Synonymous Codon Ordering: A Subtle but Prevalent Strategy of Bacteria to Improve Translational Efficiency

BackgroundIn yeast coding sequences, once a particular codon has been used, subsequent occurrence of the same amino acid tends to use codons sharing the same tRNA. Such a phenomenon of co-tRNA codons pairing bias (CTCPB) is also found in some other eukaryotes but it is not known whether it occurs in prokaryotes.Methodology/Principal FindingsIn this study, we focused on a total of 773 bacterial genomes to investigate their synonymous codon pairing preferences. After calculating the actual frequencies of synonymous codon pairs and comparing them with their expected values, we detected an obvious pairing bias towards identical codon pairs. This seems consistent with the previously reported CTCPB phenomenon, since identical codons are certainly read by the same tRNA. However, among co-tRNA but non-identical codon pairs, only 22 were often found overrepresented, suggesting that many co-tRNA codons actually do not preferentially pair together in prokaryotes. Therefore, the previously reported co-tRNA codons pairing rule needs to be more rigorously defined. The affinity differences between a tRNA anticodon and its readable codons should be taken into account. Moreover, both within-gene-shuffling tests and phylogenetic analyses support the idea that translational selection played an important role in shaping the observed synonymous codon pairing pattern in prokaryotes.ConclusionsOverall, a high level of synonymous codon pairing bias was detected in 73% investigated bacterial species, suggesting the synonymous codon ordering strategy has been prevalently adopted by prokaryotes to improve their translational efficiencies. The findings in this study also provide important clues to better understand the complex dynamics of translational process.

The Role of KRAS Mutational Analysis to Determine the Site of Origin of Metastatic Carcinoma to the Lung: A Case Report

Metastatic carcinomas involving the lung are a common specimen encountered in surgical pathology. These metastases may have different morphologic, and architectural patterns and may mimic primary pulmonary adenocarcinoma, especially the intra-alveolar (lepidic) pattern of spread which may simulate a primary pulmonary bronchioloalveolar carcinoma (adenocarcinoma in situ). We present the case of a metastatic pancreatic adenocarcinoma that morphologically mimicked bronchioloalveolar carcinoma of the lung in that the tumor had an exclusive intra-alveolar pattern of spread and had an immunophenotype that was noninformative as to the site of origin (cytokeratin 7+, cytokeratin 20−, TTF-1−). In this case, we used KRAS gene mutation analysis to support that the lung carcinoma represented a metastatic pancreatic carcinoma as they both possessed identical codon 12 KRAS mutations. We show that this method may be a useful way to prove site of origin of metastatic carcinoma—particularly if standard morphologic or immunohistochemical analysis is not definitive.

Wobble base-pairing slows in vivo translation elongation in metazoans

In the universal genetic code, most amino acids can be encoded by multiple trinucleotide codons, and the choice among available codons can influence position-specific translation elongation rates. By using sequence-based ribosome profiling, we obtained transcriptome-wide profiles of in vivo ribosome occupancy as a function of codon identity in Caenorhabditis elegans and human cells. Particularly striking in these profiles was a universal trend of higher ribosome occupancy for codons translated via G:U wobble base-pairing compared with synonymous codons that pair with the same tRNA family using G:C base-pairing. These data support a model in which ribosomal translocation is slowed at wobble codon positions.

Plant-type mitochondrial RNA editing in the protist Naegleria gruberi: FIGURE 1.

RNA editing converts hundreds of cytidines into uridines in plant mitochondrial and chloroplast transcripts. Recognition of the RNA editing sites in the organelle transcriptomes requires numerous specific, nuclear-encoded RNA-binding pentatricopeptide repeat (PPR) proteins with characteristic carboxy-terminal protein domain extensions (E/DYW) previously thought to be unique to plants. However, a small gene family of such plant-like PPR proteins of the DYW-type was recently discovered in the genome of the protist Naegleria gruberi. This raised the possibility that plant-like RNA editing may occur in this amoeboflagellate. Accordingly, we have investigated the mitochondrial transcriptome of Naegleria gruberi and here report on identification of two sites of C-to-U RNA editing in the cox1 gene and in the cox3 gene, both of which reconstitute amino acid codon identities highly conserved in evolution. An estimated 1.5 billion years of evolution separate the heterolobosean protist Naegleria from the plant lineage. The new findings either suggest horizontal gene transfer of RNA editing factors or that plant-type RNA editing is evolutionarily much more ancestral than previously thought and yet to be discovered in many other ancient eukaryotic lineages.

Unveiling the structural basis for translational ambiguity tolerance in a human fungal pathogen

In a restricted group of opportunistic fungal pathogens the universal leucine CUG codon is translated both as serine (97%) and leucine (3%), challenging the concept that translational ambiguity has a negative impact in living organisms. To elucidate the molecular mechanisms underlying the in vivo tolerance to a nonconserved genetic code alteration, we have undertaken an extensive structural analysis of proteins containing CUG-encoded residues and solved the crystal structures of the two natural isoforms of Candida albicans seryl-tRNA synthetase. We show that codon reassignment resulted in a nonrandom genome-wide CUG redistribution tailored to minimize protein misfolding events induced by the large-scale leucine-to-serine replacement within the CTG clade. Leucine or serine incorporation at the CUG position in C. albicans seryl-tRNA synthetase induces only local structural changes and, although both isoforms display tRNA serylation activity, the leucine-containing isoform is more active. Similarly, codon ambiguity is predicted to shape the function of C. albicans proteins containing CUG-encoded residues in functionally relevant positions, some of which have a key role in signaling cascades associated with morphological changes and pathogenesis. This study provides a first detailed analysis on natural reassignment of codon identity, unveiling a highly dynamic evolutionary pattern of thousands of fungal CUG codons to confer an optimized balance between protein structural robustness and functional plasticity.

Optimal Codon Identities in Bacteria: Implications from the Conflicting Results of Two Different Methods

A correlation method was recently adopted to identify selection-favored ‘optimal’ codons from 675 bacterial genomes. Surprisingly, the identities of these optimal codons were found to track the bacterial GC content, leading to a conclusion that selection would generally shape the codon usages to the same direction as the overall mutation does. Raising several concerns, here we report a thorough comparative study on 203 well-selected bacterial species, which strongly suggest that the previous conclusion is likely an illusion. Firstly, the previous study did not preclude species that are suffering weak or no selection pressures on their codon usages. For these species, as showed in this study, the optimal codon identities are prone to be incorrect and follow GC content. Secondly, the previous study only adopted the correlation method, without considering another method to test the reliability of inferred optimal codons. Actually by definition, optimal codons can also be identified by simply comparing codon usages between high- and low-expression genes. After using both methods to identify optimal codons for the selected species, we obtained highly conflicting results, suggesting at least one method is misleading. Further we found a critical problem of correlation method at the step of calculating gene bias level. Due to a failure of accurately defining the background mutation, the problem would result in wrong optimal codon identities. In other words, partial mutational effects on codon choices were mistakenly regarded as selective influences, leading to incorrect and biased optimal codon identities. Finally, considering the translational dynamics, optimal codons identified by comparison method can be well-explained by tRNA compositions, whereas optimal codons identified by correlation method can not be. For all above reasons, we conclude that real optimal codons actually do not track the genomic GC content, and correlation method is misleading in identifying optimal codons and better be avoided.

The ribosome uses two active mechanisms to unwind messenger RNA during translation

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that slows the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to enable translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting1,2, protein expression levels3,4, ribosome localization5, and cotranslational protein folding6. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases7, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding center is greatly influenced by the G•C content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favor its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: (i) it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations toward the open state, increasing the probability for the ribosome to translocate unhindered; and (ii) it also mechanically pulls apart the mRNA single-strands of the closed junction during the conformational changes that accompany ribosome translocation. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.

Functional Significance of an Evolutionarily Conserved Alanine (GCA) Resume Codon in tmRNA in Escherichia coli

Occasionally, ribosomes stall on mRNAs prior to the completion of the polypeptide chain. In Escherichia coli and other eubacteria, tmRNA-mediated trans-translation is a major mechanism that recycles the stalled ribosomes. The tmRNA possesses a tRNA-like domain and a short mRNA region encoding a short peptide (ANDENYALAA in E. coli) followed by a termination codon. The first amino acid (Ala) of this peptide encoded by the resume codon (GCN) is highly conserved in tmRNAs in different species. However, reasons for the high evolutionary conservation of the resume codon identity have remained unclear. In this study, we show that changing the E. coli tmRNA resume codon to other efficiently translatable codons retains efficient functioning of the tmRNA. However, when the resume codon was replaced with the low-usage codons, its function was adversely affected. Interestingly, expression of tRNAs decoding the low-usage codon from plasmid-borne gene copies restored efficient utilization of tmRNA. We discuss why in E. coli, the GCA (Ala) is one of the best codons and why all codons in the short mRNA of the tmRNA are decoded by the abundant tRNAs.

Improved discrimination of melanotic schwannoma from melanocytic lesions by combined morphological and GNAQ mutational analysis

The histological differential diagnosis between melanotic schwannoma, primary leptomeningeal melanocytic lesions and cellular blue nevus can be challenging. Correct diagnosis of melanotic schwannoma is important to select patients who need clinical evaluation for possible association with Carney complex. Recently, we described the presence of activating codon 209 mutations in the GNAQ gene in primary leptomeningeal melanocytic lesions. Identical codon 209 mutations have been described in blue nevi. The aims of the present study were to (1) perform a histological review of a series of lesions (initially) diagnosed as melanotic schwannoma and analyze them for GNAQ mutations, and (2) test the diagnostic value of GNAQ mutational analysis in the differential diagnosis with leptomeningeal melanocytic lesions. We retrieved 25 cases that were initially diagnosed as melanotic schwannoma. All cases were reviewed using established criteria and analyzed for GNAQ codon 209 mutations. After review, nine cases were classified as melanotic schwannoma. GNAQ mutations were absent in these nine cases. The remaining cases were reclassified as conventional schwannoma (n = 9), melanocytoma (n = 4), blue nevus (n = 1) and lesions that could not be classified with certainty as melanotic schwannoma or melanocytoma (n = 2). GNAQ codon 209 mutations were present in 3/4 melanocytomas and the blue nevus. Including results from our previous study in leptomeningeal melanocytic lesions, GNAQ mutations were highly specific (100%) for leptomeningeal melanocytic lesions compared to melanotic schwannoma (sensitivity 43%). We conclude that a detailed analysis of morphology combined with GNAQ mutational analysis can aid in the differential diagnosis of melanotic schwannoma with leptomeningeal melanocytic lesions.

Multifactorial Determinants of Protein Expression in Prokaryotic Open Reading Frames

A quantitative description of the relationship between protein expression levels and open reading frame (ORF) nucleotide sequences is important for understanding natural systems, designing synthetic systems, and optimizing heterologous expression. Codon identity, mRNA secondary structure, and nucleotide composition within ORFs markedly influence expression levels. Bioinformatic analysis of ORF sequences in 816 bacterial genomes revealed that these features show distinct regional trends. To investigate their effects on protein expression, we designed 285 synthetic genes and determined corresponding expression levels in vitro using Escherichia coli extracts. We developed a mathematical function, parameterized using this synthetic gene data set, which enables computation of protein expression levels from ORF nucleotide sequences. In addition to its practical application in the design of heterologous expression systems, this equation provides mechanistic insight into the factors that control translation efficiency. We found that expression is strongly dependent on the presence of high AU content and low secondary structure in the ORF 5′ region. Choice of high-frequency codons contributes to a lesser extent. The 3′ terminal AU content makes modest, but detectable contributions. We present a model for the effect of these factors on the three phases of ribosomal function: initiation, elongation, and termination.

General Rules for Optimal Codon Choice

Different synonymous codons are favored by natural selection for translation efficiency and accuracy in different organisms. The rules governing the identities of favored codons in different organisms remain obscure. In fact, it is not known whether such rules exist or whether favored codons are chosen randomly in evolution in a process akin to a series of frozen accidents. Here, we study this question by identifying for the first time the favored codons in 675 bacteria, 52 archea, and 10 fungi. We use a number of tests to show that the identified codons are indeed likely to be favored and find that across all studied organisms the identity of favored codons tracks the GC content of the genomes. Once the effect of the genomic GC content on selectively favored codon choice is taken into account, additional universal amino acid specific rules governing the identity of favored codons become apparent. Our results provide for the first time a clear set of rules governing the evolution of selectively favored codon usage. Based on these results, we describe a putative scenario for how evolutionary shifts in the identity of selectively favored codons can occur without even temporary weakening of natural selection for codon bias.

Selection on Codon Bias

In a wide variety of organisms, synonymous codons are used with different frequencies, a phenomenon known as codon bias. Population genetic studies have shown that synonymous sites are under weak selection and that codon bias is maintained by a balance between selection, mutation, and genetic drift. It appears that the major cause for selection on codon bias is that certain preferred codons are translated more accurately and/or efficiently. However, additional and sometimes maybe even contradictory selective forces appear to affect codon usage as well. In this review, we discuss the current understanding of the ways in which natural selection participates in the creation and maintenance of codon bias. We also raise several open questions: (i) Is natural selection weak independently of the level of codon bias? It is possible that selection for preferred codons is weak only when codon bias approaches equilibrium and may be quite strong on genes with codon bias levels that are much lower and/or above equilibrium. (ii) What determines the identity of the major codons? (iii) How do shifts in codon bias occur? (iv) What is the exact nature of selection on codon bias? We discuss these questions in depth and offer some ideas on how they can be addressed using a combination of computational and experimental analyses.

UORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

In a minority of eukaryotic mRNAs, a small functional upstream ORF (uORF), often performing a regulatory role, precedes the translation start site for the main product(s). Here, conserved uORFs in numerous ornithine decarboxylase homologs are identified from yeast to mammals. Most have noncanonical evolutionarily conserved start codons, the main one being AUU, which has not been known as an initiator for eukaryotic chromosomal genes. The AUG-less uORF present in mouse antizyme inhibitor, one of the ornithine decarboxylase homologs in mammals, mediates polyamine-induced repression of the downstream main ORF. This repression is part of an autoregulatory circuit, and one of its sensors is the AUU codon, which suggests that translation initiation codon identity is likely used for regulation in eukaryotes.

A-to-I editing sites are a genomically encoded G: Implications for the evolutionary significance and identification of novel editing sites

Ribonucleic acid (RNA) editing can extend transcriptomic and proteomic diversity by changing the identity of a particular codon. Genetic recoding as a result of adenosine-to-inosine (A-to-I) RNA editing can alter highly conserved or invariant coding positions in proteins. Interestingly, examples exist in which A-to-I editing sites in one species are fixed genomically as a G in a closely related species. Phylogenetic analysis indicates that G-to-A mutations at the DNA level may be corrected by post-transcriptional A-to-I RNA editing, while in turn, the edited I (G) may be hardwired into the genome, resulting in an A-to-G mutation. We propose a model in which nuclear A-to-I RNA editing acts as an evolutionary intermediate of genetic variation. We not only provide information on the mechanism behind the evolutionary acquisition of an A-to-I RNA editing site but also demonstrate how to predict nuclear A-to-I editing sites by identifying positions where an RNA editing event would maintain the conservation of a protein relative to its homologs in other species. We identified a novel edited site in the fourth exon of the cacophony transcript coding calcium channel alpha1 and verified it experimentally.

Codon-triplet context unveils unique features of the Candida albicans protein coding genome

BackgroundThe evolutionary forces that determine the arrangement of synonymous codons within open reading frames and fine tune mRNA translation efficiency are not yet understood. In order to tackle this question we have carried out a large scale study of codon-triplet contexts in 11 fungal species to unravel associations or relationships between codons present at the ribosome A-, P- and E-sites during each decoding cycle.ResultsOur analysis unveiled high bias within the context of codon-triplets, in particular strong preference for triplets of identical codons. We have also identified a surprisingly large number of codon-triplet combinations that vanished from fungal ORFeomes. Candida albicans exacerbated these features, showed an unbalanced tRNA population for decoding its pool of codons and used near-cognate decoding for a large set of codons, suggesting that unique evolutionary forces shaped the evolution of its ORFeome.ConclusionWe have developed bioinformatics tools for large-scale analysis of codon-triplet contexts. These algorithms identified codon-triplets context biases, allowed for large scale comparative codon-triplet analysis, and identified rules governing codon-triplet context. They could also detect alterations to the standard genetic code.