Adjusting for selection on synonymous sites in estimates of evolutionary distance

Evolution at silent sites is often used to estimate the pace of selectively neutral processes or to infer differences in divergence times of genes. However, silent sites are subject to selection in favor of preferred codons, and the strength of such selection varies dramatically across genes. Here, we use the relationship between codon bias and synonymous divergence observed in four species of the genus Saccharomyces to provide a simple correction for selection on silent sites.     

Intragenic Hill-Robertson interference influences selection intensity on synonymous mutations in Drosophila

Natural selection influences synonymous mutations and synonymouscodon usage in many eukaryotes to improve the efficiency oftranslation in highly expressed genes. Recent studies of genecomposition in eukaryotes have shown that codon usage also variesindependently of expression levels, both among genes and atthe intragenic level. Here, we investigate rates of evolution(Ks) and intensity of selection (_s) on synonymous mutationsin two groups of genes that differ greatly in the length oftheir exons, but with equivalent levels of gene expression andrates of crossing-over in Drosophila melanogaster. We estimate_s using patterns of divergence and polymorphism in 50 Drosophilagenes (100 kb of coding sequence) to take into account possiblevariation in mutation trends across the genome, among genesor among codons. We show that genes with long exons exhibithigher Ks and reduced _s compared to genes with short exons.We also show that Ks and _s vary significantly across long exons,with higher Ks and reduced _s in the central region comparedto flanking regions of the same exons, hence indicating thatthe difference between genes with short and long exons can bemostly attributed to the central region of these long exons.Although amino acid composition can also play a significantrole when estimating Ks and _s, our analyses show that the differencesin Ks and _s between genes with short and long exons and acrosslong exons cannot be explained by differences in protein composition.All these results are consistent with the Interference Selection(IS) model that proposes that the Hill-Robertson (HR) effectcaused by many weakly selected mutations has detectable evolutionaryconsequences at the intragenic level in genomes with recombination.Under the IS model, exon size and exon-intron structure influencethe effectiveness of selection, with long exons showing reducedeffectiveness of selection when compared to small exons andthe central region of long exons showing reduced intensity ofselection compared to flanking coding regions. Finally, ourresults further stress the need to consider selection on synonymousmutations and its variation—among and across genes andexons—in studies of protein evolution.     

Effect of strong directional selection on weakly selected mutations at linked sites: implication for synonymous codon usage

The fixation of weakly selected mutations can be greatly influenced by strong directional selection at linked loci. Here, I investigate a two-locus model in which weakly selected, reversible mutations occur at one locus and recurrent strong directional selection occurs at the other locus. This model is analogous to selection on codon usage at synonymous sites linked to nonsynonymous sites under strong directional selection. Two approximations obtained here describe the expected frequency of the weakly selected preferred alleles at equilibrium. These approximations, as well as simulation results, show that the level of codon bias declines with an increasing rate of substitution at the strongly selected locus, as expected from the well-understood theory that selection at one locus reduces the efficacy of selection at linked loci. These solutions are used to examine whether the negative correlation between codon bias and nonsynonymous substitution rates recently observed in Drosophila can be explained by this hitchhiking effect. It is shown that this observation can be reasonably well accounted for if a large fraction of the nonsynonymous substitutions on genes in the data set are driven by strong directional selection.     

Codon usage bias: causative factors,quantification methods and genome‐wide patterns: with emphasis on insect genomes

Codon usage bias refers to the phenomenon where specific codons are used more often than other synonymous codons during translation of genes, the extent of which varies within and among species. Molecular evolutionary investigations suggest that codon bias is manifested as a result of balance between mutational and translational selection of such genes and that this phenomenon is widespread across species and may contribute to genome evolution in a significant manner. With the advent of whole‐genome sequencing of numerous species, both prokaryotes and eukaryotes, genome‐wide patterns of codon bias are emerging in different organisms. Various factors such as expression level, GC content, recombination rates, RNA stability, codon position, gene length and others (including environmental stress and population size) can influence codon usage bias within and among species. Moreover, there has been a continuous quest towards developing new concepts and tools to measure the extent of codon usage bias of genes. In this review, we outline the fundamental concepts of evolution of the genetic code, discuss various factors that may influence biased usage of synonymous codons and then outline different principles and methods of measurement of codon usage bias. Finally, we discuss selected studies performed using whole‐genome sequences of different insect species to show how codon bias patterns vary within and among genomes. We conclude with generalized remarks on specific emerging aspects of codon bias studies and highlight the recent explosion of genome‐sequencing efforts on arthropods (such as twelve Drosophila species, species of ants, honeybee, Nasonia and Anopheles mosquitoes as well as the recent launch of a genome‐sequencing project involving 5000 insects and other arthropods) that may help us to understand better the evolution of codon bias and its biological significance.     

Widespread positive selection in synonymous sites of mammalian genes

Evolution of protein sequences is largely governed by purifying selection, with a small fraction of proteins evolving under positive selection. The evolution at synonymous positions in protein-coding genes is not nearly as well understood, with the extent and types of selection remaining, largely, unclear. A statistical test to identify purifying and positive selection at synonymous sites in protein-coding genes was developed. The method compares the rate of evolution at synonymous sites (Ks) to that in intron sequences of the same gene after sampling the aligned intron sequences to mimic the statistical properties of coding sequences. We detected purifying selection at synonymous sites in approximately 28% of the 1,562 analyzed orthologous genes from mouse and rat, and positive selection in approximately 12% of the genes. Thus, the fraction of genes with readily detectable positive selection at synonymous sites is much greater than the fraction of genes with comparable positive selection at nonsynonymous sites, i.e., at the level of the protein sequence. Unlike other genes, the genes with positive selection at synonymous sites showed no correlation between Ks and the rate of evolution in nonsynonymous sites (Ka), indicating that evolution of synonymous sites under positive selection is decoupled from protein evolution. The genes with purifying selection at synonymous sites showed significant anticorrelation between Ks and expression level and breadth, indicating that highly expressed genes evolve slowly. The genes with positive selection at synonymous sites showed the opposite trend, i.e., highly expressed genes had, on average, higher Ks. For the genes with positive selection at synonymous sites, a significantly lower mRNA stability is predicted compared to the genes with negative selection. Thus, mRNA destabilization could be an important factor driving positive selection in nonsynonymous sites, probably, through regulation of expression at the level of mRNA degradation and, possibly, also translation rate. So, unexpectedly, we found that positive selection at synonymous sites of mammalian genes is substantially more common than positive selection at the level of protein sequences. Positive selection at synonymous sites might act through mRNA destabilization affecting mRNA levels and translation.     

同义密码子使用偏嗜性对mRNA半衰期及翻译调控的影响北大核心CSCD

随着基因组学和转录组学在不同生物体遗传和细胞生物学领域的广泛应用,同义密码子使用的偏嗜性逐渐被接受,并且在研究生物进化与生物表型之间的深层联系时,同义密码子使用模式受到相关领域研究人员的重视。信使RNA(messenger RNA,mRNA)最终表达出具有正常生物活性的蛋白产物是生命活动的重要环节。被称为“第二遗传密码”的同义密码子使用模式,可以通过精微调控翻译选择压力等分子机制,从转录调控、翻译调控及代谢活动等水平表达其承载的遗传信息。研究表明,mRNA半衰期的长短对mRNA活性以及转录和翻译过程有显著的影响。因此,系统地归纳同义密码子使用模式在基因转录、翻译调控及翻译后修饰等生命活动中所扮演的角色,将有助于全方位审视生物体如何巧妙利用密码子使用模式所产生的遗传效应来精准合成不同种类蛋白质,并以此保障生长或分化的特定基因表达程序顺利执行、维持正常的生命周期。     

同义密码子通过精微翻译选择机制实现对基因的表达调控

自然界中,同义密码子的存在使得众多氨基酸能够同时被多种密码子编码合成。随着研究的深入,同义密码子使用偏嗜性发挥出的生物学功能已经渗透到了基因复制、转录、翻译以及化学修饰等生命活动过程中。基于同义密码子使用偏嗜性的生物学特性,陆续发现密码子对(codon pair)和密码子共现(codon co-occurrence)同样在使用模式上存在明显的偏嗜性。在基因表达的过程中,针对编码序列的密码子优化能够显著提升基因的表达水平,这在生物工程领域对于蛋白表达有着重要的生物学意义。此外,同义密码子使用模式在调控基因转录、化学修饰以及翻译过程中间接控制着细胞内生命活动的有序性。而这些与同义密码子使用模式有着千丝万缕联系的生命过程主要是受精微翻译选择压力来调控运行的。本文中,我们结合当前同义密码子使用模式介导的精微翻译选择压力,简述密码子使用模式如何从转录、化学修饰以及翻译等方面来影响基因表达及蛋白产物生物学功能。这将为今后生物工程学领域如何优化蛋白高效表达以及深入研究重要生物学活动中基因表达调控提供可参考的思路与理念。     

转座因子对水稻同义密码子使用偏性的影响

利用635个包含完整转座因子插入的粳稻CDS序列,对转座因子如何影响基因编码区的碱基组成及基因的表达水平,进而对基因同义密码子的使用偏性产生影响进行了详细分析。结果表明:转座因子插入极显著地影响到基因编码区的同义密码子使用但并非唯一因素;转座因子对不同基因的表达水平具有多重影响,有的基因表达被抑制,有的反而增强,但总的来说它减少了基因表达水平对同义密码子使用的影响程度。     

Patterns of selection on synonymous and nonsynonymous variants in Drosophila miranda

We have investigated patterns of within-species polymorphism and between-species divergence for synonymous and nonsynonymous variants at a set of autosomal and X-linked loci of Drosophila miranda. D. pseudoobscura and D. affinis were used for the between-species comparisons. The results suggest the action of purifying selection on nonsynonymous, polymorphic variants. Among synonymous polymorphisms, there is a significant excess of synonymous mutations from preferred to unpreferred codons and of GC to AT mutations. There was no excess of GC to AT mutations among polymorphisms at noncoding sites. This suggests that selection is acting to maintain the use of preferred codons. Indirect evidence suggests that biased gene conversion in favor of GC base pairs may also be operating. The joint intensity of selection and biased gene conversion, in terms of the product of effective population size and the sum of the selection and conversion coefficients, was estimated to be approximately 0.65.     

Nucleotide polymorphism in the RpII215 gene region of the insular species Drosophila guanche: reduced efficacy of weak selection on synonymous variation

An approximately 6.9-kb region encompassing the RpII215 gene was sequenced for 24 individuals of the island endemic species Drosophila guanche. The comparative analysis of synonymous polymorphism and divergence in D. guanche and D. subobscura, two species with pronounced differences in population size, allows contrasting the nearly neutral character of synonymous mutations. In D. guanche, unlike in D. subobscura, (1) the ratio of preferred to unpreferred synonymous changes was similar for polymorphic and fixed changes, (2) the numbers of preferred and unpreferred changes, both polymorphic and fixed, could be explained by the mutational process, and (3) the estimated scaled selection coefficient for unpreferred mutations did not differ significantly from zero. Additionally, the comparative analysis revealed that both the ratio of preferred to unpreferred synonymous changes and the frequency spectrum of unpreferred polymorphic mutations differed significantly between species. All these results indicate that a large fraction of synonymous mutations in the RpII215 gene behave as effectively neutral in D. guanche, whereas they are weakly selected in D. subobscura. The reduced efficacy of selection in the insular species constitutes strong evidence of the nearly neutral character of synonymous mutations and, therefore, of the role of weak selection in maintaining codon bias.     

Codon volatility as an indicator of positive selection: data from eukaryotic genome comparisons

It has been suggested that codon volatility (the proportion of the point-mutation neighbors of a codon that encode different amino acids) can be used as an index of past positive selection. We compared codon volatility with patterns of synonymous and nonsynonymous nucleotide substitution in genome-wide comparisons of orthologous genes between three pairs of related genomes: (1) the protists Plasmodium falciparum and P. yoelii, (2) the fungi Saccharomyces cerevisiae and S. paradoxus, and (3) the mammals mouse and rat. Codon volatility was not consistently associated with an elevated rate of nonsynonymous substitution, as would be expected under positive selection. Rather, the most consistent and powerful correlate of elevated codon volatility was nucleotide content at the second codon position, as expected, given the nature of the genetic code.     

Incorporation of transition to transversion ratio and nonsense mutations,improves the estimation of the number of synonymous and non-synonymous sites in codons

A common approach to estimate the strength and direction of selection acting on protein coding sequences is to calculate the dN/dS ratio. The method to calculate dN/dS has been widely used by many researchers and many critical reviews have been made on its application after the proposition by Nei and Gojobori in 1986. However, the method is still evolving considering the non-uniform substitution rates and pretermination codons. In our study of SNPs in 586 genes across 156 Escherichia coli strains, synonymous polymorphism in 2-fold degenerate codons were higher in comparison to that in 4-fold degenerate codons, which could be attributed to the difference between transition (Ti) and transversion (Tv) substitution rates where the average rate of a transition is four times more than that of a transversion in general. We considered both the Ti/Tv ratio, and nonsense mutation in pretermination codons, to improve estimates of synonymous (S) and non-synonymous (NS) sites. The accuracy of estimating dN/dS has been improved by considering the Ti/Tv ratio and nonsense substitutions in pretermination codons. We showed that applying the modified approach based on Ti/Tv ratio and pretermination codons results in higher values of dN/dS in 29 common genes of equal reading-frames between E. coli and Salmonella enterica. This study emphasizes the robustness of amino acid composition with varying codon degeneracy, as well as the pretermination codons when calculating dN/dS values.     

Evidence for a trade-off between translational efficiency and splicing regulation in determining synonymous codon usage in Drosophila melanogaster

In Drosophila melanogaster, synonymous codons corresponding to the most abundant cognate tRNAs are used more frequently, especially in highly expressed genes. Increased use of such "optimal" codons is considered an adaptation for translational efficiency. Need it always be the case that selection should favor the use of a translationally optimal codon? Here, we investigate one possible confounding factor, namely, the need to specify information in exons necessary to enable correct splicing. As expected from such a model, in Drosophila many codons show different usage near intron-exon boundaries versus exon core regions. However, this finding is in principle also consistent with Hill-Robertson effects modulating usage of translationally optimal codons. However, several results support the splice model over the translational selection model: 1) the trends in codon usage are strikingly similar to those in mammals in which codon usage near boundaries correlates with abundance in exonic splice enhancers (ESEs), 2) codons preferred near boundaries tend to be enriched for A and avoid C (conversely those avoided near boundaries prefer C rather than A), as expected were ESEs involved, and 3) codons preferred near boundaries are typically not translationally optimal. We conclude that usage of translationally optimal codons usage is compromised in the vicinity of splice junctions in intron-containing genes, to the effect that we observe higher levels of usage of translationally optimal codons at the center of exons. On the gene level, however, controlling for known correlates of codon bias, the impact on codon usage patterns is quantitatively small. These results have implications for inferring aspects of the mechanism of splicing given nothing more than a well-annotated genome.     

Site-to-site variation of synonymous substitution rates

We develop a new model for studying the molecular evolution of protein-coding DNA sequences. In contrast to existing models, we incorporate the potential for site-to-site heterogeneity of both synonymous and nonsynonymous substitution rates. We demonstrate that within-gene heterogeneity of synonymous substitution rates appears to be common. Using the new family of models, we investigate the utility of a variety of new statistical inference procedures, and we pay particular attention to issues surrounding the detection of sites undergoing positive selection. We discuss how failure to model synonymous rate variation in the model can lead to misidentification of sites as positively selected.     

The Codon Statistics Database: A Database of Codon Usage Bias

We present the Codon Statistics Database, an online database that contains codon usage statistics for all the species with reference or representative genomes in RefSeq (over 15,000). The user can search for any species and access two sets of tables. One set lists, for each codon, the frequency, the Relative Synonymous Codon Usage, and whether the codon is preferred. Another set of tables lists, for each gene, its GC content, Effective Number of Codons, Codon Adaptation Index, and frequency of optimal codons. Equivalent tables can be accessed for (1) all nuclear genes, (2) nuclear genes encoding ribosomal proteins, (3) mitochondrial genes, and (4) chloroplast genes (if available in the relevant assembly). The user can also search for any taxonomic group (e.g., “primates”) and obtain a table comparing all the species in the group. The database is free to access without registration at http://codonstatsdb.unr.edu.     

Experimental reduction of codon bias in the Drosophila alcohol dehydrogenase gene results in decreased ethanol tolerance of adult flies

The ethanol tolerance of adult transgenic flies of Drosophila containing between zero and ten unpreferred synonymous mutations that reduced codon bias in the alcohol dehydrogenase (Adh) gene was assayed. As the amino acid sequences of the ADH protein were identical in the four genotypes assayed, differences in ethanol tolerance were due to differences in the abundance of ADH protein, presumably driven by the effects of codon bias on translational efficiency. The ethanol tolerance of genotypes decreased with the number of unpreferred synonymous mutations, and a positive correlation between ADH protein abundance and ethanol tolerance was observed. This work confirms that the fitness effects of unpreferred synonymous mutations that reduce codon bias in a highly expressed gene are experimentally measurable in Drosophila melanogaster.     

Translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts

Codon usage in chloroplasts is different from that in prokaryotic and eukaryotic nuclear genomes. However, no experimental approach has been made to analyse the translation efficiency of individual codons in chloroplasts. We devised an in vitro assay for translation efficiencies using synthetic mRNAs, and measured the translation efficiencies of five synonymous codon groups in tobacco chloroplasts. Among four alanine codons (GCN, where N is U, C, A or G), GCU was the most efficient for translation, whereas the chloroplast genome lacks tRNA genes corresponding to GCU. Phenylalanine and tyrosine are each encoded by two codons (UUU/C and UAU/C, respectively). Phenylalanine UUC and tyrosine UAC were translated more than twice as efficiently than UUU and UAU, respectively, contrary to their codon usage, whereas translation efficiencies of synonymous codons for alanine, aspartic acid and asparagine were parallel to their codon usage. These observations indicate that translation efficiencies of individual codons are not always correlated with codon usage in vitro in chloroplasts. This raises an important issue for foreign gene expression in chloroplasts.     

Stress induced MAPK genes show distinct pattern of codon usage in Arabidopsis thaliana,Glycine max and Oryza sativa

Mitogen activated protein kinase (MAPK) genes provide resistance to various biotic and abiotic stresses. Codon usage profiling of the genes reveals the characteristic features of the genes like nucleotide composition, gene expressivity, optimal codons etc. The present study is a comparative analysis of codon usage patterns for different MAPK genes in three organisms, viz. Arabidopsis thaliana, Glycine max (soybean) and Oryza sativa (rice). The study has revealed a high AT content in MAPK genes of Arabidopsis and soybean whereas in rice a balanced AT-GC content at the third synonymous position of codon. The genes show a low bias in codon usage profile as reflected in the higher values (50.83 to 56.55) of effective number of codons (N_c). The prediction of gene expression profile in the MAPK genes revealed that these genes might be under the selective pressure of translational optimization as reflected in the low codon adaptation index (CAI) values ranging from 0.147 to 0.208.     

Genetic robustness and selection at the protein level for synonymous codons

Synonymous codons are neutral at the protein level, therefore natural selection at the protein level should have no effect on their frequencies. Synonymous codons, however, differ in their capacity to reduce the effects of errors: after mutation, certain codons keep on coding for the same amino acid or for amino acids with similar properties, while other synonymous codons produce very different amino acids. Therefore, the impact of errors on a coding sequence (genetic robustness) can be measured by analysing its codon usage. I analyse the codon usage of sequenced nuclear and cytoplasmic genomes and I show that there is an extensive variation in genetic robustness at the DNA sequence level, both among genomes and among genes of the same genome. I also show theoretically that robustness can be adaptive, that is natural selection may lead to a preference for codons that reduce the impact of errors. If selection occurs only among the mutants of a codon (e.g. among the progeny before the adult phase), however, the codons that are more sensitive to the effects of mutations may increase in frequency because they manage to get rid more easily of deleterious mutations. I also suggest other possible explanations for the evolution of genetic robustness at the codon level.