Evolutionary divergence of exon flanks: a dissection of mutability and selection

The intronic sequences flanking exon-intron junctions (i.e., exon flanks) are important for splice site recognition and pre-mRNA splicing. Recent studies show a higher degree of sequence conservation at flanks of alternative exons, compared to flanks of constitutive exons. In this article we performed a detailed analysis on the evolutionary divergence of exon flanks between human and chimpanzee, aiming to dissect the impact of mutability and selection on their evolution. Inside exon flanks, sites that might reside in ancestral CpG dinucleotides evolved significantly faster than sites outside of ancestral CpG dinucleotides. This result reflects a systematic variation of mutation rates (mutability) at exon flanks, depending on the local CpG contexts. Remarkably, we observed a significant reduction of the nucleotide substitution rate in flanks of alternatively spliced exons, independent of the site-by-site variation in mutability due to different CpG contexts. Our data provide concrete evidence for increased purifying selection at exon flanks associated with regulation of alternative splicing.     

Evidence of selectively driven codon usage in rice: implications for GC content evolution of Gramineae genes

Two gene classes characterized by high and low GC content have been found in rice and other cereals, but not dicot genomes. We used paralogs with high and low GC contents in rice and found: (a) a greater increase in GC content at exonic fourfold-redundant sites than at flanking introns; (b) with reference to their orthologs in Arabidopsis, most substitution sites between the two kinds of paralogs are found at 2- and 4-degenerate sites with a T-->C mode, while A-->C and A-->G play major roles at 0-degenerate sites; and (c) high-GC genes have greater bias and codon usage is skewed toward codons that are preferred in highly expressed genes. We believe this is strong evidence for selectively driven codon usage in rice. Another cereal, maize, also showed the same trend as in rice. This represents a potential evolutionary process for the origin of genes with a high GC content in rice and other cereals.     

Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer.

Cytosine methylation at CpG dinucleotides is thought to cause more than one-third of all transition mutations responsible for human genetic diseases and cancer. We investigated the methylation status of the CpG dinucleotide at codon 248 in exon 7 of the p53 gene because this codon is a hot spot for inactivating mutations in the germ line and in most human somatic tissues examined. Codon 248 is contained within an HpaII site (CCGG), and the methylation status of this and flanking CpG sites was analyzed by using the methylation-sensitive enzymes CfoI (GCGC) and HpaII. Codon 248 and the CfoI and HpaII sites in the flanking introns were methylated in every tissue and cell line examined, indicating extensive methylation of this region in the p53 gene. Exhaustive treatment of an osteogenic sarcoma cell line, TE85, with the hypomethylating drug 5-aza-2'-deoxycytidine did not demethylate codon 248 or the CfoI sites in intron 6, although considerable global demethylation of the p53 gene was induced. Constructs containing either exon 7 alone or exon 7 and the flanking introns were transfected into TE85 cells to determine whether de novo methylation would occur. The presence of exon 7 alone caused some de novo methylation to occur at codon 248. More extensive de novo methylation of the CfoI sites in intron 6, which contains an Alu sequence, occurred in cells transfected with a vector containing exon 7 and flanking introns. With longer time in culture, there was increased methylation at the CfoI sites, and de novo methylation of codon 248 and its flanking HpaII sites was observed. These de novo-methylated sites were also resistant to 5-aza-2'-deoxycytidine-induced demethylation. The frequent methylation of codon 248 and adjacent Alu sequence may explain the enhanced mutability of this site as a result of the deamination of the 5-methylcytosine.     

Evolution of base composition in the insulin and insulin-like growth factor genes

The genomes of homeothermic (warm-blooded) vertebrates are mosaic interspersions of homogeneously GC-rich and GC-poor regions (isochores). Evolution of genome compartmentalization and GC-rich isochores is hypothesized to reflect either selective advantages of an elevated GC content or chromosome location and mutational pressure associated with the timing of DNA replication in germ cells. To address the present controversy regarding the origins and maintenance of isochores in homeothermic vertebrates, newly obtained as well as published nucleotide sequences of the insulin and insulin-like growth factor (IGF) genes, members of a well-characterized gene family believed to have evolved by repeated duplication and divergence, were utilized to examine the evolution of base composition in nonconstrained (flanking) and weakly constrained (introns and fourfold degenerate sites) regions. A phylogeny derived from amino acid sequences supports a common evolutionary history for the insulin/IGF family genes. In cold- blooded vertebrates, insulin and the IGFs were similar in base composition. In contrast, insulin and IGF-II demonstrate dramatic increases in GC richness in mammals, but no such trend occurred in IGF- I. Base composition of the coding portions of the insulin and IGF genes across vertebrates correlated (r = 0.90) with that of the introns and flanking regions. The GC content of homologous introns differed dramatically between insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level of noncoding regions in neighboring genes. Our findings suggest that the base composition of introns and flanking regions is determined by chromosomal location and the mutational pressure of the isochore in which the sequences are embedded. An elevated GC content at codon third positions in the insulin and the IGF genes may reflect selective constraints on the usage of synonymous codons.      

Evolution of noncoding and silent coding sites in the Plasmodium falciparum and Plasmodium reichenowi genomes

We compared levels of sequence divergence between fourfold synonymous coding sites and noncoding sites from the intergenic and intronic regions of the Plasmodium falciparum and Plasmodium reichenowi genomes. We observed significant differences in the level of divergence between these classes of silent sites. Fourfold synonymous coding sites exhibited the highest level of sequence divergence, followed by introns, and then intergenic sequences. This pattern of relative divergence rates has been observed in primate genomes but was unexpected in Plasmodium due to a paucity of variation at silent sites in P. falciparum and the corollary hypothesis that silent sites in this genome may be subject to atypical selective constraints. Exclusion of hypermutable CpG dinucleotides reduces the divergence level of synonymous coding sites to that of intergenic sites but does not diminish the significantly higher divergence level of introns relative to intergenic sites. A greater than expected incidence of CpG dinucleotides in intergenic regions less than 500 bp from genes may indicate selective maintenance of regulatory motifs containing CpGs. Divergence rates of different classes of silent sites in these Plasmodium genomes are determined by a combination of mutational and selective pressures.     

Codon usage bias covaries with expression breadth and the rate of synonymous evolution in humans, but this is not evidence for selection.

In numerous species, from bacteria to Drosophila, evidence suggests that selection acts even on synonymous codon usage: codon bias is greater in more abundantly expressed genes, the rate of synonymous evolution is lower in genes with greater codon bias, and there is consistency between genes in the same species in which codons are preferred. In contrast, in mammals, while nonequal use of alternative codons is observed, the bias is attributed to the background variance in nucleotide concentrations, reflected in the similar nucleotide composition of flanking noncoding and exonic third sites. However, a systematic examination of the covariants of codon usage controlling for background nucleotide content has yet to be performed. Here we present a new method to measure codon bias that corrects for background nucleotide content and apply this to 2396 human genes. Nearly all (99%) exhibit a higher amount of codon bias than expected by chance. The patterns associated with selectively driven codon bias are weakly recovered: Broadly expressed genes have a higher level of bias than do tissue-specific genes, the bias is higher for genes with lower rates of synonymous substitutions, and certain codons are repeatedly preferred. However, while these patterns are suggestive, the first two patterns appear to be methodological artifacts. The last pattern reflects in part biases in usage of nucleotide pairs. We conclude that we find no evidence for selection on codon usage in humans.     

应用统计方法确定真核基因外显子

研究了人类基因外显子、内含子的编码结构及D值,发现外显子、内含子间有显著不同,应用“剪接法”进行外显子定位,成功率达到74.6％,表明这一方法可在实际研究中应用。     

Alternatively and constitutively spliced exons are subject to different evolutionary forces

There has been a controversy on whether alternatively spliced exons (ASEs) evolve faster than constitutively spliced exons (CSEs). Although it has been noted that ASEs are subject to weaker selective constraints than CSEs, so they evolve faster, there have also been studies that indicated slower evolution in ASEs than in CSEs. In this study, we retrieve more than 5,000 human-mouse orthologous exons and calculate the synonymous (KS) and nonsynonymous (KA) substitution rates in these exons. Our results show that ASEs have higher KA values and higher KA/KS ratios than CSEs, indicating faster amino acid-level evolution in ASEs. The faster evolution may be in part due to weaker selective constraints. It is also possible that the faster rate is in part due to faster functional evolution in ASEs. On the other hand, the majority of ASEs have lower KS values than CSEs. With reference to the substitution rate in introns, we show that the KS values in ASEs are close to the neutral substitution rate, whereas the synonymous substitution rate in CSEs has likely been accelerated. The elevated synonymous rate in CSEs is not related to CpG dinucleotides or low-complexity regions of protein but may be weakly related to codon usage bias. The overall trends of higher KA and lower KS in ASEs than in CSEs are also observed in human-rat and mouse-rat comparisons. Therefore, our observations hold for mammals of different molecular clocks.     

Intronic alternative splicing regulators identified by comparative genomics in nematodes

Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high- scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene.     

Selection on Silent Sites in the Rodent H3 Histone Gene Family

Selection promoting differential use of synonymous codons has been shown for several unicellular organisms and for Drosophila, but not for mammals. Selection coefficients operating on synonymous codons are likely to be extremely small, so that a very large effective population size is required for selection to overcome the effects of drift. In mammals, codon-usage bias is believed to be determined exclusively by mutation pressure, with differences between genes due to large-scale variation in base composition around the genome. The replication-dependent histone genes are expressed at extremely high levels during periods of DNA synthesis, and thus are among the most likely mammalian genes to be affected by selection on synonymous codon usage. We suggest that the extremely biased pattern of codon usage in the H3 genes is determined in part by selection. Silent site G + C content is much higher than expected based on flanking sequence G + C content, compared to other rodent genes with similar silent site base composition but lower levels of expression. Dinucleotide-mediated mutation bias does affect codon usage, but the affect is limited to the choice between G and C in some fourfold degenerate codons. Gene conversion between the two clusters of histone genes has not been an important force in the evolution of the H3 genes, but gene conversion appears to have had some effect within the cluster on chromosome 13.     

Genomic DNA sequence of Rhesus (M. mulatta) cystic fibrosis (CFTR) gene

Cystic fibrosis is a common human genetic disease caused by mutations in CFTR, a gene that codes for a chloride channel that is regulated by phosphorylation and cytosolic nucleotides. As part of a program to discover natural animal models for human genetic diseases, we have determined the genomic sequence of CFTR in the Rhesus monkey, Macaca mulatta. The coding region of rhesus CFTR is 98.3% identical to human CFTR at the nucleotide level and 98.2% identical and 99.7% similar at the amino acid level. Partial sequences of flanking introns (5582 base pair positions analyzed) revealed 91.1% identity with human introns. Relative to rhesus intronic sequence, the human sequences had 27 insertions and 22 deletions. Primer sequences for amplification of rhesus genomic CFTR sequences are provided. The accession number is AF013753 (all 27 exons and some flanking intronic sequence). Received: 27 August 1992 / Accepted: 5 December 1997     

A test of translational selection at 'silent' sites in the human genome: base composition comparisons in alternatively spliced genes

Natural selection appears to discriminate among synonymous codons to enhance translational efficiency in a wide range of prokaryotes and eukaryotes. Codon bias is strongly related to gene expression levels in these species. In addition, between-gene variation in silent DNA divergence is inversely correlated with codon bias. However, in mammals, between-gene comparisons are complicated by distinctive nucleotide-content bias (isochores) throughout the genome. In this study, we attempted to identify translational selection by analyzing the DNA sequences of alternatively spliced genes in humans and in Drosophila melanogaster. Among codons in an alternatively spliced gene, those in constitutively expressed exons are translated more often than those in alternatively spliced exons. Thus, translational selection should act more strongly to bias codon usage and reduce silent divergence in constitutive than in alternative exons. By controlling for regional forces affecting base-composition evolution, this within-gene comparison makes it possible to detect codon selection at synonymous sites in mammals. We found that GC-ending codons are more abundant in constitutive than alternatively spliced exons in both Drosophila and humans. Contrary to our expectation, however, silent DNA divergence between mammalian species is higher in constitutive than in alternative exons.     

The elevated GC content at exonic third sites is not evidence against neutralist models of isochore evolution

The human genome is divided into isochores, large stretches (>300 kb) of genomic DNA with more or less consistent GC content. Mutational/neutralist and selectionist models have been put forward to explain their existence. A major criticism of the mutational models is that they cannot account for the higher GC content at fourfold-redundant silent sites within exons (GC4) than in flanking introns (GCi). Indeed, it has been asserted that it is hard to envisage a mutational bias explanation, as it is difficult to see how repair enzymes might act differently in exons and their flanking introns. However, this rejection, we note, ignores the effects of transposable elements (TEs), which are a major component of introns and tend to cause them to have a GC content different from (usually lower than) that dictated by point mutational processes alone. As TEs tend not to insert at the extremities of introns, this model predicts that GC content at the extremities of introns should be more like that at GC4 than are the intronic interiors. This we show to be true. The model also correctly predicts that small introns should have a composition more like that at GC4 than large introns. We conclude that the logic of the previous rejection of neutralist models is unsafe.     

The emergence of alternative 3' and 5' splice site exons from constitutive exons

Alternative 3' and 5' splice site (ss) events constitute a significant part of all alternative splicing events. These events were also found to be related to several aberrant splicing diseases. However, only few of the characteristics that distinguish these events from alternative cassette exons are known currently. In this study, we compared the characteristics of constitutive exons, alternative cassette exons, and alternative 3'ss and 5'ss exons. The results revealed that alternative 3'ss and 5'ss exons are an intermediate state between constitutive and alternative cassette exons, where the constitutive side resembles constitutive exons, and the alternative side resembles alternative cassette exons. The results also show that alternative 3'ss and 5'ss exons exhibit low levels of symmetry (frame-preserving), similar to constitutive exons, whereas the sequence between the two alternative splice sites shows high symmetry levels, similar to alternative cassette exons. In addition, flanking intronic conservation analysis revealed that exons whose alternative splice sites are at least nine nucleotides apart show a high conservation level, indicating intronic participation in the regulation of their splicing, whereas exons whose alternative splice sites are fewer than nine nucleotides apart show a low conservation level. Further examination of these exons, spanning seven vertebrate species, suggests an evolutionary model in which the alternative state is a derivative of an ancestral constitutive exon, where a mutation inside the exon or along the flanking intron resulted in the creation of a new splice site that competes with the original one, leading to alternative splice site selection. This model was validated experimentally on four exons, showing that they indeed originated from constitutive exons that acquired a new competing splice site during evolution.     

Evidence for purifying selection against synonymous mutations in mammalian exonic splicing enhancers

Silent sites in mammals have classically been assumed to be free from selective pressures. Consequently, the synonymous substitution rate (Ks) is often used as a proxy for the mutation rate. Although accumulating evidence demonstrates that the assumption is not valid, the mechanism by which selection acts remain unclear. Recent work has revealed that the presence of exonic splicing enhancers (ESEs) in coding sequence might influence synonymous evolution. ESEs are predominantly located near intron-exon junctions, which may explain the reduced single-nucleotide polymorphism (SNP) density in these regions. Here we show that synonymous sites in putative ESEs evolve more slowly than the remaining exonic sequence. Differential mutabilities of ESEs do not appear to explain this difference. We observe that substitution frequency at fourfold synonymous sites decreases as one approaches the ends of exons, consistent with the existing SNP data. This gradient is at least in part explained by ESEs being more abundant near junctions. Between-gene variation in Ks is hence partly explained by the proportion of the gene that acts as an ESE. Given the relative abundance of ESEs and the reduced rates of synonymous divergence within them, we estimate that constraints on synonymous evolution within ESEs causes the true mutation rate to be underestimated by not more than approximately 8%. We also find that Ks outside of ESEs is much lower in alternatively spliced exons than in constitutive exons, implying that other causes of selection on synonymous mutations exist. Additionally, selection on ESEs appears to affect nonsynonymous sites and may explain why amino acid usage near intron-exon junctions is nonrandom.