Analysis of evolution of exon-intron structure of eukaryotic genes

The availability of multiple, complete eukaryotic genome sequences allows one to address many fundamental evolutionary questions on genome scale. One such important, long-standing problem is evolution of exon-intron structure of eukaryotic genes. Analysis of orthologous genes from completely sequenced genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists. The data on shared and lineage-specific intron positions were used as the starting point for evolutionary reconstruction with parsimony and maximum-likelihood approaches. Parsimony methods produce reconstructions with intron-rich ancestors but also infer lineage-specific, in many cases, high levels of intron loss and gain. Different probabilistic models gave opposite results, apparently depending on model parameters and assumptions, from domination of intron loss, with extremely intron-rich ancestors, to dramatic excess of gains, to the point of denying any true conservation of intron positions among deep eukaryotic lineages. Development of models with adequate, realistic parameters and assumptions seems to be crucial for obtaining more definitive estimates of intron gain and loss in different eukaryotic lineages. Many shared intron positions were detected in ancestral eukaryotic paralogues which evolved by duplication prior to the divergence of extant eukaryotic lineages. These findings indicate that numerous introns were present in eukaryotic genes already at the earliest stages of evolution of eukaryotes and are compatible with the hypothesis that the original, catastrophic intron invasion accompanied the emergence of the eukaryotic cells. Comparison of various features of old and younger introns starts shedding light on probable mechanisms of intron insertion, indicating that propagation of old introns is unlikely to be a major mechanism for origin of new ones. The existence and structure of ancestral protosplice sites were addressed by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and, accordingly, are not subject to selection for splicing efficiency. It was shown that introns indeed predominantly insert into or are fixed in specific protosplice sites which have the consensus sequence (A/C)AG|Gt.     

Analysis of Fungal Genomes Reveals Commonalities of Intron Gain or Loss and Functions in Intron-Poor Species

Previous evolutionary reconstructions have concluded that early eukaryotic ancestors including both the last common ancestor of eukaryotes and of all fungi had intron-rich genomes. By contrast, some extant eukaryotes have few introns, underscoring the complex histories of intron–exon structures, and raising the question as to why these few introns are retained. Here, we have used recently available fungal genomes to address a variety of questions related to intron evolution. Evolutionary reconstruction of intron presence and absence using 263 diverse fungal species supports the idea that massive intron reduction through intron loss has occurred in multiple clades. The intron densities estimated in various fungal ancestors differ from zero to 7.6 introns per 1 kb of protein-coding sequence. Massive intron loss has occurred not only in microsporidian parasites and saccharomycetous yeasts, but also in diverse smuts and allies. To investigate the roles of the remaining introns in highly-reduced species, we have searched for their special characteristics in eight intron-poor fungi. Notably, the introns of ribosome-associated genes RPL7 and NOG2 have conserved positions; both intron-containing genes encoding snoRNAs. Furthermore, both the proteins and snoRNAs are involved in ribosome biogenesis, suggesting that the expression of the protein-coding genes and noncoding snoRNAs may be functionally coordinated. Indeed, these introns are also conserved in three-quarters of fungi species. Our study shows that fungal introns have a complex evolutionary history and underappreciated roles in gene expression.     

Widespread intron loss suggests retrotransposon activity in ancient apicomplexans

Several facets of spliceosomal intron in apicomplexans remain mysterious. First, intron numbers vary across species by 2 orders of magnitude, indicating massive intron loss and/or gain. Second, previous studies have shown very different evolutionary patterns over different timescales, with 100-fold higher rates of intron loss/gain between genera than within genera. Third, the timing and dynamics of nearly complete intron loss in Cryptosporidium species, as well as reasons for retention of the few remaining introns, remain unknown. We compared intron positions in 785 orthologous genes between 3 moderate to intron-rich apicomplexan species. We estimate that the Theileria-Plasmodium ancestor had 4.5 times as many introns as modern Plasmodium species and 38% more than modern Theileria species, and that subsequent intron losses have outnumbered intron gains by 5.8 to 1 in Theileria and by some 56 to 1 in Plasmodium. Several patterns suggest that these intron losses occurred by recombination with reverse-transcribed mRNAs. Intriguingly, this finding suggests significant retrotransposon activity in the lineages leading to both Theileria and Plasmodium, in contrast to the dearth of known retrotransposons and intron loss within modern species from both genera. We also compared genomes from Cryptosporidium parvum and C. hominis and found no evidence of ongoing intron loss, nor of intron gain. By contrast, Cryptosporidium introns are less evolutionary conserved with Toxoplasma than are introns from other apicomplexans; thus the few remaining introns are not simply indispensable ancestral introns.     

Origin and evolution of spliceosomal introns

ABSTRACT: Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded 'introns first' held that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns. Thus, the introns-first scenario is not supported by any evidence but exon-intron structure of protein-coding genes appears to have evolved concomitantly with the eukaryotic cell, and introns were a major factor of evolution throughout the history of eukaryotes. This article was reviewed by I. King Jordan, Manuel Irimia (nominated by Anthony Poole), Tobias Mourier (nominated by Anthony Poole), and Fyodor Kondrashov. For the complete reports, see the Reviewers' Reports section.     

Conservation versus parallel gains in intron evolution

Orthologous genes from distant eukaryotic species, e.g. animals and plants, share up to 25–30% intron positions. However, the relative contributions of evolutionary conservation and parallel gain of new introns into this pattern remain unknown. Here, the extent of independent insertion of introns in the same sites (parallel gain) in orthologous genes from phylogenetically distant eukaryotes is assessed within the framework of the protosplice site model. It is shown that protosplice sites are no more conserved during evolution of eukaryotic gene sequences than random sites. Simulation of intron insertion into protosplice sites with the observed protosplice site frequencies and intron densities shows that parallel gain can account but for a small fraction (5–10%) of shared intron positions in distantly related species. Thus, the presence of numerous introns in the same positions in orthologous genes from distant eukaryotes, such as animals, fungi and plants, appears to reflect mostly bona fide evolutionary conservation.     

Evolutionary convergence on highly-conserved 3' intron structures in intron-poor eukaryotes and insights into the ancestral eukaryotic genome

The presence of spliceosomal introns in eukaryotes raises a range of questions about genomic evolution. Along with the fundamental mysteries of introns' initial proliferation and persistence, the evolutionary forces acting on intron sequences remain largely mysterious. Intron number varies across species from a few introns per genome to several introns per gene, and the elements of intron sequences directly implicated in splicing vary from degenerate to strict consensus motifs. We report a 50-species comparative genomic study of intron sequences across most eukaryotic groups. We find two broad and striking patterns. First, we find that some highly intron-poor lineages have undergone evolutionary convergence to strong 3' consensus intron structures. This finding holds for both branch point sequence and distance between the branch point and the 3' splice site. Interestingly, this difference appears to exist within the genomes of green alga of the genus Ostreococcus, which exhibit highly constrained intron sequences through most of the intron-poor genome, but not in one much more intron-dense genomic region. Second, we find evidence that ancestral genomes contained highly variable branch point sequences, similar to more complex modern intron-rich eukaryotic lineages. In addition, ancestral structures are likely to have included polyT tails similar to those in metazoans and plants, which we found in a variety of protist lineages. Intriguingly, intron structure evolution appears to be quite different across lineages experiencing different types of genome reduction: whereas lineages with very few introns tend towards highly regular intronic sequences, lineages with very short introns tend towards highly degenerate sequences. Together, these results attest to the complex nature of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron structures across modern lineages, and the impressive evolutionary malleability of eukaryotic gene structures.     

Prevalence of intron gain over intron loss in the evolution of paralogous gene families

The mechanisms and evolutionary dynamics of intron insertion and loss in eukaryotic genes remain poorly understood. Reconstruction of parsimonious scenarios of gene structure evolution in paralogous gene families in animals and plants revealed numerous gains and losses of introns. In all analyzed lineages, the number of acquired new introns was substantially greater than the number of lost ancestral introns. This trend held even for lineages in which vertical evolution of genes involved more intron losses than gains, suggesting that gene duplication boosts intron insertion. However, dating gene duplications and the associated intron gains and losses based on the molecular clock assumption showed that very few, if any, introns were gained during the last ~100 million years of animal and plant evolution, in agreement with previous conclusions reached through analysis of orthologous gene sets. These results are generally compatible with the emerging notion of intensive insertion and loss of introns during transitional epochs in contrast to the relative quiet of the intervening evolutionary spans.     

Evidence of splice signal migration from exon to intron during intron evolution

A comparison of the nucleotide sequences around the splice junctions that flank old (shared by two or more major lineages of eukaryotes) and new (lineage-specific) introns in eukaryotic genes reveals substantial differences in the distribution of information between introns and exons. Old introns have a lower information content in the exon regions adjacent to the splice sites than new introns but have a corresponding higher information content in the intron itself. This suggests that introns insert into nonrandom (proto-splice) sites but, during the evolution of an intron after insertion, the splice signal shifts from the flanking exon regions to the ends of the intron itself. Accumulation of information inside the intron during evolution suggests that new introns largely emerge de novo rather than through propagation and migration of old introns.     

A detailed history of intron-rich eukaryotic ancestors inferred from a global survey of 100 complete genomes

Protein-coding genes in eukaryotes are interrupted by introns, but intron densities widely differ between eukaryotic lineages. Vertebrates, some invertebrates and green plants have intron-rich genes, with 6-7 introns per kilobase of coding sequence, whereas most of the other eukaryotes have intron-poor genes. We reconstructed the history of intron gain and loss using a probabilistic Markov model (Markov Chain Monte Carlo, MCMC) on 245 orthologous genes from 99 genomes representing the three of the five supergroups of eukaryotes for which multiple genome sequences are available. Intron-rich ancestors are confidently reconstructed for each major group, with 53 to 74% of the human intron density inferred with 95% confidence for the Last Eukaryotic Common Ancestor (LECA). The results of the MCMC reconstruction are compared with the reconstructions obtained using Maximum Likelihood (ML) and Dollo parsimony methods. An excellent agreement between the MCMC and ML inferences is demonstrated whereas Dollo parsimony introduces a noticeable bias in the estimations, typically yielding lower ancestral intron densities than MCMC and ML. Evolution of eukaryotic genes was dominated by intron loss, with substantial gain only at the bases of several major branches including plants and animals. The highest intron density, 120 to 130% of the human value, is inferred for the last common ancestor of animals. The reconstruction shows that the entire line of descent from LECA to mammals was intron-rich, a state conducive to the evolution of alternative splicing.     

New maximum likelihood estimators for eukaryotic intron evolution

The evolution of spliceosomal introns remains poorly understood. Although many approaches have been used to infer intron evolution from the patterns of intron position conservation, the results to date have been contradictory. In this paper, we address the problem using a novel maximum likelihood method, which allows estimation of the frequency of intron insertion target sites, together with the rates of intron gain and loss. We analyzed the pattern of 10,044 introns (7,221 intron positions) in the conserved regions of 684 sets of orthologs from seven eukaryotes. We determined that there is an average of one target site per 11.86 base pairs (bp) (95% confidence interval, 9.27 to 14.39 bp). In addition, our results showed that: (i) overall intron gains are ~25% greater than intron losses, although specific patterns vary with time and lineage; (ii) parallel gains account for ~18.5% of shared intron positions; and (iii) reacquisition following loss accounts for ~0.5% of all intron positions. Our results should assist in resolving the long-standing problem of inferring the evolution of spliceosomal introns.     

Patterns of intron gain and conservation in eukaryotic genes

Background:  

The presence of introns in protein-coding genes is a universal feature of eukaryotic genome organization, and the genes of multicellular eukaryotes, typically, contain multiple introns, a substantial fraction of which share position in distant taxa, such as plants and animals. Depending on the methods and data sets used, researchers have reached opposite conclusions on the causes of the high fraction of shared introns in orthologous genes from distant eukaryotes. Some studies conclude that shared intron positions reflect, almost entirely, a remarkable evolutionary conservation, whereas others attribute it to parallel gain of introns. To resolve these contradictions, it is crucial to analyze the evolution of introns by using a model that minimally relies on arbitrary assumptions.     

Remarkable interkingdom conservation of intron positions and massive,lineage-specific intron loss and gain in eukaryotic evolution

Sequencing of eukaryotic genomes allows one to address major evolutionary problems, such as the evolution of gene structure. We compared the intron positions in 684 orthologous gene sets from 8 complete genomes of animals, plants, fungi, and protists and constructed parsimonious scenarios of evolution of the exon-intron structure for the respective genes. Approximately one-third of the introns in the malaria parasite Plasmodium falciparum are shared with at least one crown group eukaryote; this number indicates that these introns have been conserved through >1.5 billion years of evolution that separate Plasmodium from the crown group. Paradoxically, humans share many more introns with the plant Arabidopsis thaliana than with the fly or nematode. The inferred evolutionary scenario holds that the common ancestor of Plasmodium and the crown group and, especially, the common ancestor of animals, plants, and fungi had numerous introns. Most of these ancestral introns, which are retained in the genomes of vertebrates and plants, have been lost in fungi, nematodes, arthropods, and probably Plasmodium. In addition, numerous introns have been inserted into vertebrate and plant genes, whereas, in other lineages, intron gain was much less prominent.     

The biology of intron gain and loss

Intron density in eukaryote genomes varies by more than three orders of magnitude, so there must have been extensive intron gain and/or intron loss during evolution. A favored and partial explanation for this range of intron densities has been that introns have accumulated stochastically in large eukaryote genomes during their evolution from an intron-poor ancestor. However, recent studies have shown that some eukaryotes lost many introns, whereas others accumulated and/or gained many introns. In this article, we discuss the growing evidence that these differences are subject to selection acting on introns depending on the biology of the organism and the gene involved.     

Extremely intron-rich genes in the alveolate ancestors inferred with a flexible maximum-likelihood approach

Chromalveolates are a large, diverse supergroup of unicellulareukaryotes that includes Apicomplexa, dinoflagellates, ciliates(three lineages that form the alveolate branch), heterokonts,haptophytes, and cryptomonads (three lineages comprising thechromist branch). All sequenced genomes of chromalveolates haverelatively low intron density in protein-coding genes, and fewintron positions are shared between chromalveolate lineages.In contrast, genes of different chromalveolates share many intronpositions with orthologous genes from other eukaryotic supergroups,in particular, the intron-rich orthologs from animals and plants.Reconstruction of the history of intron gain and loss duringthe evolution of chromalveolates using a general and flexiblemaximum-likelihood approach indicates that genes of the ancestorsof chromalveolates and, particularly, alveolates had unexpectedlyhigh intron densities. It is estimated that the chromalveolateancestor had, approximately, two-third of the human intron density,whereas the intron density in the genes of the alveolate ancestoris estimated to be slightly greater than the human intron density.Accordingly, it is inferred that the evolution of chromalveolateswas dominated by intron loss. The conclusion that ancestralchromalveolate forms had high intron densities is unexpectedbecause all extant unicellular eukaryotes have relatively fewintrons and are thought to be unable to maintain numerous intronsdue to intense purifying selection in their, typically, largepopulations. It is suggested that, at early stages of evolution,chromalveolates went through major population bottlenecks thatwere accompanied by intron invasion.     

Analysis of ribosomal protein gene structures: implications for intron evolution

Many spliceosomal introns exist in the eukaryotic nuclear genome. Despite much research, the evolution of spliceosomal introns remains poorly understood. In this paper, we tried to gain insights into intron evolution from a novel perspective by comparing the gene structures of cytoplasmic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs), which are held to be of archaeal and bacterial origin, respectively. We analyzed 25 homologous pairs of CRP and MRP genes that together had a total of 527 intron positions. We found that all 12 of the intron positions shared by CRP and MRP genes resulted from parallel intron gains and none could be considered to be “conserved,” i.e., descendants of the same ancestor. This was supported further by the high frequency of proto-splice sites at these shared positions; proto-splice sites are proposed to be sites for intron insertion. Although we could not definitively disprove that spliceosomal introns were already present in the last universal common ancestor, our results lend more support to the idea that introns were gained late. At least, our results show that MRP genes were intronless at the time of endosymbiosis. The parallel intron gains between CRP and MRP genes accounted for 2.3% of total intron positions, which should provide a reliable estimate for future inferences of intron evolution.     

Near intron positions are reliable phylogenetic markers: an application to holometabolous insects

Today, the reconstruction of the organismal evolutionary tree is based mainly on molecular sequence data. However, sequence data are sometimes insufficient to reliably resolve in particular deep branches. Thus, it is highly desirable to find novel, more reliable types of phylogenetic markers that can be derived from the wealth of genomic data. Here, we consider the gain of introns close to older preexisting ones. Because correct splicing is impeded by very small exons, nearby pairs of introns very rarely coexist, that is, the gain of the new intron is nearly always associated with the loss of the old intron. Both events may even be directly connected as in cases of intron migration. Therefore, it should be possible to identify one of the introns as ancient (plesiomorphic) and the other as novel (derived or apomorphic). To test the suitability of such near intron pairs (NIPs) as a marker class for phylogenetic analysis, we undertook an analysis of the evolutionary positions of bees and wasps (Hymenoptera) and beetles (Coleoptera) in relation to moths (Lepidoptera) and dipterans (Diptera) using recently completed genome project data. By scanning 758 putatively orthologous gene structures of Apis mellifera (Hymenoptera) and Tribolium castaneum (Coleoptera), we identified 189 pairs of introns, one from each species, which are located less than 50 nt from each other. A comparison with genes from 5 other holometabolan and 9 metazoan outgroup genomes resulted in 22 shared derived intron positions found in beetle as well as in butterflies and/or dipterans. This strongly supports a basal position of hymenopterans in the holometabolous insect tree. In addition, we found 31 and 12 intron positions apomorphic for A. mellifera and T. castaneum, respectively, which seem to represent changes inside these branches. Another 12 intron pairs indicate parallel intron gains or extraordinarily small exons. In conclusion, we show here that the analysis of phylogenetically nested, nearby intron pairs is suitable to identify evolutionarily younger intron positions and to determine their relative age, which should be of equal importance for the understanding of intron evolution and the reconstruction of the eukaryotic tree.