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1.  The origin of introns and their role in eukaryogenesis: a compromise solution to the introns-early versus introns-late debate? 
Biology Direct  2006;1:22.
Ever since the discovery of 'genes in pieces' and mRNA splicing in eukaryotes, origin and evolution of spliceosomal introns have been considered within the conceptual framework of the 'introns early' versus 'introns late' debate. The 'introns early' hypothesis, which is closely linked to the so-called exon theory of gene evolution, posits 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. Under this scenario, the absence of spliceosomal introns in prokaryotes is considered to be a result of "genome streamlining". The 'introns late' hypothesis counters that spliceosomal introns emerged only in eukaryotes, and moreover, have been inserted into protein-coding genes continuously throughout the evolution of eukaryotes. Beyond the formal dilemma, the more substantial side of this debate has to do with possible roles of introns in the evolution of eukaryotes.
I argue that several lines of evidence now suggest a coherent solution to the introns-early versus introns-late debate, and the emerging picture of intron evolution integrates aspects of both views although, formally, there seems to be no support for the original version of introns-early. Firstly, there is growing evidence that spliceosomal introns evolved from group II self-splicing introns which are present, usually, in small numbers, in many bacteria, and probably, moved into the evolving eukaryotic genome from the α-proteobacterial progenitor of the mitochondria. Secondly, the concept of a primordial pool of 'virus-like' genetic elements implies that self-splicing introns are among the most ancient genetic entities. Thirdly, reconstructions of the ancestral state of eukaryotic genes suggest that the last common ancestor of extant eukaryotes had an intron-rich genome. Thus, it appears that ancestors of spliceosomal introns, indeed, have existed since the earliest stages of life's evolution, in a formal agreement with the introns-early scenario. However, there is no evidence that these ancient introns ever became widespread before the emergence of eukaryotes, hence, the central tenet of introns-early, the role of introns in early evolution of proteins, has no support. However, the demonstration that numerous introns invaded eukaryotic genes at the outset of eukaryotic evolution and that subsequent intron gain has been limited in many eukaryotic lineages implicates introns as an ancestral feature of eukaryotic genomes and refutes radical versions of introns-late. Perhaps, most importantly, I argue that the intron invasion triggered other pivotal events of eukaryogenesis, including the emergence of the spliceosome, the nucleus, the linear chromosomes, the telomerase, and the ubiquitin signaling system. This concept of eukaryogenesis, in a sense, revives some tenets of the exon hypothesis, by assigning to introns crucial roles in eukaryotic evolutionary innovation.
The scenario of the origin and evolution of introns that is best compatible with the results of comparative genomics and theoretical considerations goes as follows: self-splicing introns since the earliest stages of life's evolution – numerous spliceosomal introns invading genes of the emerging eukaryote during eukaryogenesis – subsequent lineage-specific loss and gain of introns. The intron invasion, probably, spawned by the mitochondrial endosymbiont, might have critically contributed to the emergence of the principal features of the eukaryotic cell. This scenario combines aspects of the introns-early and introns-late views.
this article was reviewed by W. Ford Doolittle, James Darnell (nominated by W. Ford Doolittle), William Martin, and Anthony Poole.
PMCID: PMC1570339  PMID: 16907971
2.  A Detailed History of Intron-rich Eukaryotic Ancestors Inferred from a Global Survey of 100 Complete Genomes 
PLoS Computational Biology  2011;7(9):e1002150.
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.
Author Summary
In eukaryotes, protein-coding genes are interrupted by non-coding introns. The intron densities widely differ, from 6–7 introns per kilobase of coding sequence in vertebrates, some invertebrates and plants, to only a few introns across the entire genome in many unicellular forms. We applied a robust statistical methodology, Markov Chain Monte Carlo, to reconstruct the history of intron gain and loss throughout the evolution of eukaryotes using a set of 245 homologous genes from 99 genomes that represent the diversity of eukaryotes. Intron-rich ancestors were confidently inferred for each major eukaryotic group including 53% to 74% of the human intron density for the last eukaryotic common ancestor, and 120% to 130% of the human value for the last common ancestor of animals. Evolution of eukaryotic genes involved primarily intron loss, with substantial gain only at the bases of several major branches including plants and animals. Thus, the common ancestor of all extant eukaryotes was a complex organism with a gene architecture resembling those in multicellular organisms. The line of descent from the last common ancestor to mammals was an uninterrupted intron-rich state that, given the error-prone splicing in intron-rich organisms, was conducive to the elaboration of functional alternative splicing.
PMCID: PMC3174169  PMID: 21935348
3.  Analysis of Ribosomal Protein Gene Structures: Implications for Intron Evolution  
PLoS Genetics  2006;2(3):e25.
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.
Genes in eukaryotes are usually intervened by extra bits of DNA sequence, called introns, that have to be removed after the genes are transcribed into RNA. Why do introns exist in eukaryotic genes? What is the reason for the increased intron density in higher eukaryotes? There is much that is not known about introns. This research tries to clarify the evolutionary process by which introns arose by comparing the gene structures of two types of ribosomal proteins; one in cytoplasm and the other in mitochondria of the cell. Since cytoplasm and mitochondria are of archaeal and bacterial origin, respectively, cytoplasmic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) are believed to diverge at the same time with the divergence of archaea and bacteria. Thus, a comparative analysis of CRP and MRP genes may reveal whether introns already existed at the last common ancestor of archaea and bacteria (introns-early) or whether they emerged late (introns-late). The results make it clear, at least, that all of the introns in MRP genes were gained during the course of eukaryotic evolution and therefore lend more support to the introns-late theory.
PMCID: PMC1386722  PMID: 16518464
4.  Comparative genomics of eukaryotic small nucleolar RNAs reveals deep evolutionary ancestry amidst ongoing intragenomic mobility 
Small nucleolar (sno)RNAs are required for posttranscriptional processing and modification of ribosomal, spliceosomal and messenger RNAs. Their presence in both eukaryotes and archaea indicates that snoRNAs are evolutionarily ancient. The location of some snoRNAs within the introns of ribosomal protein genes has been suggested to belie an RNA world origin, with the exons of the earliest protein-coding genes having evolved around snoRNAs after the advent of templated protein synthesis. Alternatively, this intronic location may reflect more recent selection for coexpression of snoRNAs and ribosomal components, ensuring rRNA modification by snoRNAs during ribosome synthesis. To gain insight into the evolutionary origins of this genetic organization, we examined the antiquity of snoRNA families and the stability of their genomic location across 44 eukaryote genomes.
We report that dozens of snoRNA families are traceable to the Last Eukaryotic Common Ancestor (LECA), but find only weak similarities between the oldest eukaryotic snoRNAs and archaeal snoRNA-like genes. Moreover, many of these LECA snoRNAs are located within the introns of host genes independently traceable to the LECA. Comparative genomic analyses reveal the intronic location of LECA snoRNAs is not ancestral however, suggesting the pattern we observe is the result of ongoing intragenomic mobility. Analysis of human transcriptome data indicates that the primary requirement for hosting intronic snoRNAs is a broad expression profile. Consistent with ongoing mobility across broadly-expressed genes, we report a case of recent migration of a non-LECA snoRNA from the intron of a ubiquitously expressed non-LECA host gene into the introns of two LECA genes during the evolution of primates.
Our analyses show that snoRNAs were a well-established family of RNAs at the time when eukaryotes began to diversify. While many are intronic, this association is not evolutionarily stable across the eukaryote tree; ongoing intragenomic mobility has erased signal of their ancestral gene organization, and neither introns-first nor evolved co-expression adequately explain our results. We therefore present a third model — constrained drift — whereby individual snoRNAs are intragenomically mobile and may occupy any genomic location from which expression satisfies phenotype.
PMCID: PMC3511168  PMID: 22978381
snoRNA; Last Eukaryotic Common Ancestor; Intron; Retrotransposition; Introns-first; Constrained drift
5.  Comparative genomic analysis of fungal genomes reveals intron-rich ancestors 
Genome Biology  2007;8(10):R223.
Analysis of intron gain and loss in fungal genomes provides support for an intron-rich fungus-animal ancestor.
Eukaryotic protein-coding genes are interrupted by spliceosomal introns, which are removed from transcripts before protein translation. Many facets of spliceosomal intron evolution, including age, mechanisms of origins, the role of natural selection, and the causes of the vast differences in intron number between eukaryotic species, remain debated. Genome sequencing and comparative analysis has made possible whole genome analysis of intron evolution to address these questions.
We analyzed intron positions in 1,161 sets of orthologous genes across 25 eukaryotic species. We find strong support for an intron-rich fungus-animal ancestor, with more than four introns per kilobase, comparable to the highest known modern intron densities. Indeed, the fungus-animal ancestor is estimated to have had more introns than any of the extant fungi in this study. Thus, subsequent fungal evolution has been characterized by widespread and recurrent intron loss occurring in all fungal clades. These results reconcile three previously proposed methods for estimation of ancestral intron number, which previously gave very different estimates of ancestral intron number for eight eukaryotic species, as well as a fourth more recent method. We do not find a clear inverse correspondence between rates of intron loss and gain, contrary to the predictions of selection-based proposals for interspecific differences in intron number.
Our results underscore the high intron density of eukaryotic ancestors and the widespread importance of intron loss through eukaryotic evolution.
PMCID: PMC2246297  PMID: 17949488
6.  Sm/Lsm Genes Provide a Glimpse into the Early Evolution of the Spliceosome 
PLoS Computational Biology  2009;5(3):e1000315.
The spliceosome, a sophisticated molecular machine involved in the removal of intervening sequences from the coding sections of eukaryotic genes, appeared and subsequently evolved rapidly during the early stages of eukaryotic evolution. The last eukaryotic common ancestor (LECA) had both complex spliceosomal machinery and some spliceosomal introns, yet little is known about the early stages of evolution of the spliceosomal apparatus. The Sm/Lsm family of proteins has been suggested as one of the earliest components of the emerging spliceosome and hence provides a first in-depth glimpse into the evolving spliceosomal apparatus. An analysis of 335 Sm and Sm-like genes from 80 species across all three kingdoms of life reveals two significant observations. First, the eukaryotic Sm/Lsm family underwent two rapid waves of duplication with subsequent divergence resulting in 14 distinct genes. Each wave resulted in a more sophisticated spliceosome, reflecting a possible jump in the complexity of the evolving eukaryotic cell. Second, an unusually high degree of conservation in intron positions is observed within individual orthologous Sm/Lsm genes and between some of the Sm/Lsm paralogs. This suggests that functional spliceosomal introns existed before the emergence of the complete Sm/Lsm family of proteins; hence, spliceosomal machinery with considerably fewer components than today's spliceosome was already functional.
Author Summary
The spliceosome is a complex molecular machine that removes intervening sequences (introns) from mRNAs. It is unique to eukaryotes. Although prokaryotes have self-splicing introns, they completely lack spliceosomal introns and the spliceosome itself. Yet even the simplest eukaryotic organisms have introns and a rather complex spliceosomal apparatus. Little is known about how this amazing machine rapidly evolved in early eukaryotes. Here, we attempt to reconstruct a part of this evolutionary process using one of the most fundamental components of the spliceosome—the Sm and Lsm family of proteins. Using sequence and structure analysis as well as the analysis of the intron positions in Sm and Lsm genes in conjunction with a wealth of published data, we propose a plausible scenario for some aspects of spliceosomal evolution. In particular, we suggest that the Lsm family of genes could have been the first and the most essential component that allowed rudimentary splicing of early spliceosomal introns. Extensive duplications of Lsm genes and the later rise of the Sm gene family likely reflect a gradual increase in complexity of the spliceosome.
PMCID: PMC2650416  PMID: 19282982
7.  Mechanisms Used for Genomic Proliferation by Thermophilic Group II Introns 
PLoS Biology  2010;8(6):e1000391.
Studies of mobile group II introns from a thermophilic cyanobacterium reveal how these introns proliferate within genomes and might explain the origin of introns and retroelements in higher organisms.
Mobile group II introns, which are found in bacterial and organellar genomes, are site-specific retroelments hypothesized to be evolutionary ancestors of spliceosomal introns and retrotransposons in higher organisms. Most bacteria, however, contain no more than one or a few group II introns, making it unclear how introns could have proliferated to higher copy numbers in eukaryotic genomes. An exception is the thermophilic cyanobacterium Thermosynechococcus elongatus, which contains 28 closely related copies of a group II intron, constituting ∼1.3% of the genome. Here, by using a combination of bioinformatics and mobility assays at different temperatures, we identified mechanisms that contribute to the proliferation of T. elongatus group II introns. These mechanisms include divergence of DNA target specificity to avoid target site saturation; adaptation of some intron-encoded reverse transcriptases to splice and mobilize multiple degenerate introns that do not encode reverse transcriptases, leading to a common splicing apparatus; and preferential insertion within other mobile introns or insertion elements, which provide new unoccupied sites in expanding non-essential DNA regions. Additionally, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on elevated temperatures to help promote DNA strand separation, enabling access to a larger number of DNA target sites by base pairing of the intron RNA, with minimal constraint from the reverse transcriptase. Our results provide insight into group II intron proliferation mechanisms and show that higher temperatures, which are thought to have prevailed on Earth during the emergence of eukaryotes, favor intron proliferation by increasing the accessibility of DNA target sites. We also identify actively mobile thermophilic introns, which may be useful for structural studies, gene targeting in thermophiles, and as a source of thermostable reverse transcriptases.
Author Summary
Group II introns are bacterial mobile elements thought to be ancestors of introns and retroelements in higher organisms. They comprise a catalytically active intron RNA and an intron-encoded reverse transcriptase, which promotes splicing of the intron from precursor RNA and integration of the excised intron into new genomic sites. While most bacteria have small numbers of group II introns, in the thermophilic cyanobacterium Thermosynechococcus elongatus, a single intron has proliferated and constitutes 1.3% of the genome. Here, we investigated how the T. elongatus introns proliferated to such high copy numbers. We found divergence of DNA target specificity, evolution of reverse transcriptases that splice and mobilize multiple degenerate introns, and preferential insertion into other mobile introns or insertion elements, which provide new integration sites in non-essential regions of the genome. Further, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on higher temperatures to help promote DNA strand separation, facilitating access to DNA target sites. We speculate how these mechanisms, including elevated temperature, might have contributed to intron proliferation in early eukaryotes. We also identify actively mobile thermophilic introns, which may be useful for structural studies and biotechnological applications.
PMCID: PMC2882425  PMID: 20543989
8.  Intronization, de-intronization and intron sliding are rare in Cryptococcus 
Eukaryotic pre-mRNA gene transcripts are processed by the spliceosome to remove portions of the transcript, called spliceosomal introns. The spliceosome recognizes intron boundaries by the presence of sequence signals (motifs) contained in the actual transcript, thus sequence changes in the genome that affect existing splicing signals or create new signals may lead to changes in transcript splicing patterns. Such changes may lead to previously excluded (intronic) transcript regions being included (exonic) or vice versa. Such changes can affect the encoded protein sequence and/or post-transcriptional regulation, and are thus a potentially important source of genomic and phenotypic novelty. Two recent papers suggest that such changes may be a major force in remodeling of eukaryotic gene structures, however the rate of occurrence of such changes has not been assessed at the genomic level.
I studied four closely related species of Cryptoccocus fungi. Among 28,256 studied introns, canonical GT/C...AG boundaries are nearly universally conserved across all four species. Among only 40 observed cases of cDNA-confirmed non-conserved intron boundaries, most are likely to involve alternative splicing. I find only five cases of "intronization," intron creation from an internal exonic region by de novo emergence of new splicing boundaries, and no cases of the reverse process, "de-intronization." I find no more than ten clear cases of true movement of an intron boundary of a possibly constitutively spliced intron, and no clear cases of true "intron sliding," in which changes in the positions of both intron boundaries could lead to a movement of the intron position along the coding sequence.
These results suggest that intronization, de-intronization, and intron boundary movement are rare events in evolution.
PMCID: PMC2740785  PMID: 19664208
9.  Recurrent Loss of Specific Introns during Angiosperm Evolution 
PLoS Genetics  2014;10(12):e1004843.
Numerous instances of presence/absence variations for introns have been documented in eukaryotes, and some cases of recurrent loss of the same intron have been suggested. However, there has been no comprehensive or phylogenetically deep analysis of recurrent intron loss. Of 883 cases of intron presence/absence variation that we detected in five sequenced grass genomes, 93 were confirmed as recurrent losses and the rest could be explained by single losses (652) or single gains (118). No case of recurrent intron gain was observed. Deep phylogenetic analysis often indicated that apparent intron gains were actually numerous independent losses of the same intron. Recurrent loss exhibited extreme non-randomness, in that some introns were removed independently in many lineages. The two larger genomes, maize and sorghum, were found to have a higher rate of both recurrent loss and overall loss and/or gain than foxtail millet, rice or Brachypodium. Adjacent introns and small introns were found to be preferentially lost. Intron loss genes exhibited a high frequency of germ line or early embryogenesis expression. In addition, flanking exon A+T-richness and intron TG/CG ratios were higher in retained introns. This last result suggests that epigenetic status, as evidenced by a loss of methylated CG dinucleotides, may play a role in the process of intron loss. This study provides the first comprehensive analysis of recurrent intron loss, makes a series of novel findings on the patterns of recurrent intron loss during the evolution of the grass family, and provides insight into the molecular mechanism(s) underlying intron loss.
Author Summary
The spliceosomal introns are nucleotide sequences that interrupt coding regions of eukaryotic genes and are removed by RNA splicing after transcription. Recent studies have reported several examples of possible recurrent intron loss or gain, i.e., introns that are independently removed from or inserted into the identical sites more than once in an investigated phylogeny. However, the frequency, evolutionary patterns or other characteristics of recurrent intron turnover remain unknown. We provide results for the first comprehensive analysis of recurrent intron turnover within a plant family and show that recurrent intron loss represents a considerable portion of all intron losses identified and intron loss events far outnumber intron gain events. We also demonstrate that recurrent intron loss is non-random, affecting only a small number of introns that are repeatedly lost, and that different lineages show significantly different rates of intron loss. Our results suggest a possible role of DNA methylation in the process of intron loss. Moreover, this study provides strong support for the model of intron loss by reverse transcriptase mediated conversion of genes by their processed mRNA transcripts.
PMCID: PMC4256211  PMID: 25474210
10.  Extensive intron gain in the ancestor of placental mammals 
Biology Direct  2011;6:59.
Genome-wide studies of intron dynamics in mammalian orthologous genes have found convincing evidence for loss of introns but very little for intron turnover. Similarly, large-scale analysis of intron dynamics in a few vertebrate genomes has identified only intron losses and no gains, indicating that intron gain is an extremely rare event in vertebrate evolution. These studies suggest that the intron-rich genomes of vertebrates do not allow intron gain. The aim of this study was to search for evidence of de novo intron gain in domesticated genes from an analysis of their exon/intron structures.
A phylogenomic approach has been used to analyse all domesticated genes in mammals and chordates that originated from the coding parts of transposable elements. Gain of introns in domesticated genes has been reconstructed on well established mammalian, vertebrate and chordate phylogenies, and examined as to where and when the gain events occurred. The locations, sizes and amounts of de novo introns gained in the domesticated genes during the evolution of mammals and chordates has been analyzed. A significant amount of intron gain was found only in domesticated genes of placental mammals, where more than 70 cases were identified. De novo gained introns show clear positional bias, since they are distributed mainly in 5' UTR and coding regions, while 3' UTR introns are very rare. In the coding regions of some domesticated genes up to 8 de novo gained introns have been found. Intron densities in Eutheria-specific domesticated genes and in older domesticated genes that originated early in vertebrates are lower than those for normal mammalian and vertebrate genes. Surprisingly, the majority of intron gains have occurred in the ancestor of placentals.
This study provides the first evidence for numerous intron gains in the ancestor of placental mammals and demonstrates that adequate taxon sampling is crucial for reconstructing intron evolution. The findings of this comprehensive study slightly challenge the current view on the evolutionary stasis in intron dynamics during the last 100 - 200 My. Domesticated genes could constitute an excellent system on which to analyse the mechanisms of intron gain in placental mammals.
Reviewers: this article was reviewed by Dan Graur, Eugene V. Koonin and Jürgen Brosius.
PMCID: PMC3257199  PMID: 22112745
11.  Evolutionary Convergence on Highly-Conserved 3′ Intron Structures in Intron-Poor Eukaryotes and Insights into the Ancestral Eukaryotic Genome 
PLoS Genetics  2008;4(8):e1000148.
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.
Author Summary
The spliceosomal introns that interrupt eukaryotic genes show great number and sequence variation across species, from the rare, highly uniform yeast introns to the ubiquitous and highly variable vertebrate intron sequences. The causes of these differences remain mysterious. We studied sequences of intron branch points and 3′ termini in 50 eukaryotic species. All intron-rich species exhibit variable 3′ sequences. However, intron-poor species range from variable sequences, to uniform branch point motifs, to uniform branch point motifs in uniform positions along the intronic sequence. This is a more complex pattern than the clear relationship between intron number and 5′ intron sequence uniformity found previously. The correspondence of sequence uniformity and intron number extends to species of the green algal genus Ostreococcus, in which the single intron-rich genomic region shows far more variable intron sequences than in the otherwise intron-poor genome. We suggest that different concentrations of spliceosomal complexes may explain these differences. In addition, we report the existence of 3′ polyT tails in diverse eukaryotic protists, suggesting that this structure is ancestral. Together, these results underscore the complexity of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron sequences in modern eukaryotes, and the impressive evolutionary malleability of eukaryotic genes.
PMCID: PMC2483917  PMID: 18688272
12.  Evolutionary dynamics of U12-type spliceosomal introns 
Many multicellular eukaryotes have two types of spliceosomes for the removal of introns from messenger RNA precursors. The major (U2) spliceosome processes the vast majority of introns, referred to as U2-type introns, while the minor (U12) spliceosome removes a small fraction (less than 0.5%) of introns, referred to as U12-type introns. U12-type introns have distinct sequence elements and usually occur together in genes with U2-type introns. A phylogenetic distribution of U12-type introns shows that the minor splicing pathway appeared very early in eukaryotic evolution and has been lost repeatedly.
We have investigated the evolution of U12-type introns among eighteen metazoan genomes by analyzing orthologous U12-type intron clusters. Examination of gain, loss, and type switching shows that intron type is remarkably conserved among vertebrates. Among 180 intron clusters, only eight show intron loss in any vertebrate species and only five show conversion between the U12 and the U2-type. Although there are only nineteen U12-type introns in Drosophila melanogaster, we found one case of U2 to U12-type conversion, apparently mediated by the activation of cryptic U12 splice sites early in the dipteran lineage. Overall, loss of U12-type introns is more common than conversion to U2-type and the U12 to U2 conversion occurs more frequently among introns of the GT-AG subtype than among introns of the AT-AC subtype. We also found support for natural U12-type introns with non-canonical terminal dinucleotides (CT-AC, GG-AG, and GA-AG) that have not been previously reported.
Although complete loss of the U12-type spliceosome has occurred repeatedly, U12 introns are extremely stable in some taxa, including eutheria. Loss of U12 introns or the genes containing them is more common than conversion to the U2-type. The degeneracy of U12-type terminal dinucleotides among natural U12-type introns is higher than previously thought.
PMCID: PMC2831892  PMID: 20163699
13.  Nonsense-Mediated Decay Enables Intron Gain in Drosophila 
PLoS Genetics  2010;6(1):e1000819.
Intron number varies considerably among genomes, but despite their fundamental importance, the mutational mechanisms and evolutionary processes underlying the expansion of intron number remain unknown. Here we show that Drosophila, in contrast to most eukaryotic lineages, is still undergoing a dramatic rate of intron gain. These novel introns carry significantly weaker splice sites that may impede their identification by the spliceosome. Novel introns are more likely to encode a premature termination codon (PTC), indicating that nonsense-mediated decay (NMD) functions as a backup for weak splicing of new introns. Our data suggest that new introns originate when genomic insertions with weak splice sites are hidden from selection by NMD. This mechanism reduces the sequence requirement imposed on novel introns and implies that the capacity of the spliceosome to recognize weak splice sites was a prerequisite for intron gain during eukaryotic evolution.
Author Summary
The surprising observation 30 years ago that genes are interrupted by non-coding introns changed our view of gene architecture. Intron number varies dramatically among species; ranging from nine introns/gene in humans to less than one in some simple eukyarotes. Here we ask where new introns come from and how they are maintained in a population. We find that novel introns do not arise from pre-existing introns, although the mechanisms that generate novel introns remain unclear. We also show that novel introns carry only weak signals for their identification and removal, and therefore depend on nonsense-mediated decay (NMD). NMD maintains RNA quality control by degrading transcripts that have not been spliced properly. We propose that NMD shelters novel introns from natural selection. This increases the likelihood that a novel intron will rise in frequency and be maintained within a population, thus increasing the rate of intron gain.
PMCID: PMC2809761  PMID: 20107520
14.  Conserved intron positions in ancient protein modules 
Biology Direct  2007;2:7.
The timing of the origin of introns is of crucial importance for an understanding of early genome architecture. The Exon theory of genes proposed a role for introns in the formation of multi-exon proteins by exon shuffling and predicts the presence of conserved splice sites in ancient genes. In this study, large-scale analysis of potential conserved splice sites was performed using an intron-exon database (ExInt) derived from GenBank.
A set of conserved intron positions was found by matching identical splice sites sequences from distantly-related eukaryotic kingdoms. Most amino acid sequences with conserved introns were homologous to consensus sequences of functional domains from conserved proteins including kinases, phosphatases, small GTPases, transporters and matrix proteins. These included ancient proteins that originated before the eukaryote-prokaryote split, for instance the catalytic domain of protein phosphatase 2A where a total of eleven conserved introns were found. Using an experimental setup in which the relation between a splice site and the ancientness of its surrounding sequence could be studied, it was found that the presence of an intron was positively correlated to the ancientness of its surrounding sequence. Intron phase conservation was linked to the conservation of the gene sequence and not to the splice site sequence itself. However, no apparent differences in phase distribution were found between introns in conserved versus non-conserved sequences.
The data confirm an origin of introns deep in the eukaryotic branch and is in concordance with the presence of introns in the first functional protein modules in an 'Exon theory of genes' scenario. A model is proposed in which shuffling of primordial short exonic sequences led to the formation of the first functional protein modules, in line with hypotheses that see the formation of introns integral to the origins of genome evolution.
This article was reviewed by Scott Roy (nominated by Anthony Poole), Sandro de Souza (nominated by Manyuan Long), and Gáspár Jékely.
PMCID: PMC1800838  PMID: 17288589
15.  Exon definition as a potential negative force against intron losses in evolution 
Biology Direct  2008;3:46.
Previous studies have indicated that the wide variation in intron density (the number of introns per gene) among different eukaryotes largely reflects varying degrees of intron loss during evolution. The most popular model, which suggests that organisms lose introns through a mechanism in which reverse-transcribed cDNA recombines with the genomic DNA, concerns only one mutational force.
Using exons as the units of splicing-site recognition, exon definition constrains the length of exons. An intron-loss event results in fusion of flanking exons and thus a larger exon. The large size of the newborn exon may cause splicing errors, i.e., exon skipping, if the splicing of pre-mRNAs is initiated by exon definition. By contrast, if the splicing of pre-mRNAs is initiated by intron definition, intron loss does not matter. Exon definition may thus be a selective force against intron loss. An organism with a high frequency of exon definition is expected to experience a low rate of intron loss throughout evolution and have a high density of spliceosomal introns.
The majority of spliceosomal introns in vertebrates may be maintained during evolution not because of potential functions, but because of their splicing mechanism (i.e., exon definition). Further research is required to determine whether exon definition is a negative force in maintaining the high intron density of vertebrates.
This article was reviewed by Dr. Scott W. Roy (nominated by Dr. John Logsdon), Dr. Eugene V. Koonin, and Dr. Igor B. Rogozin (nominated by Dr. Mikhail Gelfand). For the full reviews, please go to the Reviewers' comments section.
PMCID: PMC2614967  PMID: 19014515
16.  Diverse Forms of RPS9 Splicing Are Part of an Evolving Autoregulatory Circuit 
PLoS Genetics  2012;8(3):e1002620.
Ribosomal proteins are essential to life. While the functions of ribosomal protein-encoding genes (RPGs) are highly conserved, the evolution of their regulatory mechanisms is remarkably dynamic. In Saccharomyces cerevisiae, RPGs are unusual in that they are commonly present as two highly similar gene copies and in that they are over-represented among intron-containing genes. To investigate the role of introns in the regulation of RPG expression, we constructed 16 S. cerevisiae strains with precise deletions of RPG introns. We found that several yeast introns function to repress rather than to increase steady-state mRNA levels. Among these, the RPS9A and RPS9B introns were required for cross-regulation of the two paralogous gene copies, which is consistent with the duplication of an autoregulatory circuit. To test for similar intron function in animals, we performed an experimental test and comparative analyses for autoregulation among distantly related animal RPS9 orthologs. Overexpression of an exogenous RpS9 copy in Drosophila melanogaster S2 cells induced alternative splicing and degradation of the endogenous copy by nonsense-mediated decay (NMD). Also, analysis of expressed sequence tag data from distantly related animals, including Homo sapiens and Ciona intestinalis, revealed diverse alternatively-spliced RPS9 isoforms predicted to elicit NMD. We propose that multiple forms of splicing regulation among RPS9 orthologs from various eukaryotes operate analogously to translational repression of the alpha operon by S4, the distant prokaryotic ortholog. Thus, RPS9 orthologs appear to have independently evolved variations on a fundamental autoregulatory circuit.
Author Summary
Eukaryotic genes are littered with non-coding intervening sequences, or introns, that must be precisely excised from a messenger RNA before it can be properly translated into protein. Despite their ubiquity, the evolution and function of introns remain poorly understood. Consequently, we cannot accurately predict the functions of individual introns in any organism. In this manuscript, we used a combination of comparative genomics and experimental tests to identify functional introns. First, we looked for signatures of selection to identify important introns in the model yeast Saccharomyces cerevisiae, which focused our attention on the introns of ribosomal protein genes. We then genetically deleted these introns to assess their function. Unlike mammalian introns, we found that yeast introns were not required for high levels of gene expression. Instead, particular introns (we focus on those within genes encoding ribosomal protein S9) were required to fine-tune gene expression through autoregulation. Surprisingly, animal orthologs of these genes also use introns to autoregulate through multiple forms of alternative splicing. We speculate that the introns of ribosomal protein genes, in particular, readily evolve means for autoregulation to meet the demanding requirements of ribosomal protein genes to maintain tight control of gene expression.
PMCID: PMC3315480  PMID: 22479208
17.  Intron Gains and Losses in the Evolution of Fusarium and Cryptococcus Fungi 
Genome Biology and Evolution  2012;4(11):1148-1161.
The presence of spliceosomal introns in eukaryotic genes poses a major puzzle for the study of genome evolution. Intron densities vary enormously among distant lineages. However, the mechanisms driving intron gains are poorly understood and very few intron gains and losses have been documented over short evolutionary time spans. Fungi emerged recently as excellent models to study intron evolution and “reverse splicing” was found to be a major driver of recent intron gains in a clade of ascomycete fungi. We screened a total of 38 genomes from two fungal clades important in medicine and agriculture to identify intron gains and losses both within and between species. We detected 86 and 198 variable intron positions in the Cryptococcus and Fusarium clades, respectively. Some genes underwent extensive changes in their exon–intron structure, with up to six variable intron positions per gene. We identified a very recently gained intron in a group of tomato-infecting strains belonging to the F. oxysporum species complex. In the human pathogen C. gattii, we found recent intron losses in subtypes of the species. The two studied fungal clades provided evidence for extensive changes in their exon–intron structure within and among closely related species. We show that both intronization of previously coding DNA and insertion of exogenous DNA are the major drivers of intron gains.
PMCID: PMC3514964  PMID: 23054310
spliceosomal introns; intron gains; Fusarium; Cryptococcus; population genomics
18.  Genes with a large intronic burden show greater evolutionary conservation on the protein level 
The existence of introns in eukaryotic genes is believed to provide an evolutionary advantage by increasing protein diversity through exon shuffling and alternative splicing. However, this eukaryotic feature is associated with the necessity of exclusion of intronic sequences, which requires considerable energy expenditure and can lead to splicing errors. The relationship between intronic burden and evolution is poorly understood. The goal of this study was to analyze the relationship between the intronic burden and the level of evolutionary conservation of the gene.
We found a positive correlation between the level of evolutionary conservation of a gene and its intronic burden. The level of evolutionary conservation was estimated using the conservation index (CI). The CI value was determined on the basis of the most distant ortholog of the human protein sequence and ranged from 0 (the gene was unique to the human genome) to 9 (an ortholog of the human gene was detected in plants). In multivariable model, both the number of introns and total intron size remained significant predictors of CI. We also found that the number of alternative splice variants was positively correlated with CI.
The expression level of a gene was negatively correlated with the number of introns and total size of intronic region. Genes with a greater intronic burden had lower density of missense and nonsense mutations in the coding regions of the gene, which suggests that they are under a stronger pressure from purifying selection.
We identified a positive association between intronic burden and CI. One of the possible explanations of this is the idea of a cost-benefits balance. Evolutionarily conserved (functionally important) genes can “afford” the negative consequences of maintaining multiple introns because these consequences are outweighed by the benefit of maintaining the gene. Evolutionarily conserved and functionally important genes may use introns to create novel splice variants to tune the gene function to developmental stage and tissue type.
PMCID: PMC3995522  PMID: 24629165
Exon/intron structure; Intronic burden; Evolutionary conservation
19.  Endogenous Mechanisms for the Origins of Spliceosomal Introns 
Journal of Heredity  2009;100(5):591-596.
Over 30 years since their discovery, the origin of spliceosomal introns remains uncertain. One nearly universally accepted hypothesis maintains that spliceosomal introns originated from self-splicing group-II introns that invaded the uninterrupted genes of the last eukaryotic common ancestor (LECA) and proliferated by “insertion” events. Although this is a possible explanation for the original presence of introns and splicing machinery, the emphasis on a high number of insertion events in the genome of the LECA neglects a considerable body of empirical evidence showing that spliceosomal introns can simply arise from coding or, more generally, nonintronic sequences within genes. After presenting a concise overview of some of the most common hypotheses and mechanisms for intron origin, we propose two further hypotheses that are broadly based on central cellular processes: 1) internal gene duplication and 2) the response to aberrant and fortuitously spliced transcripts. These two nonmutually exclusive hypotheses provide a powerful way to explain the establishment of spliceosomal introns in eukaryotes without invoking an exogenous source.
PMCID: PMC2877546  PMID: 19635762
group-II introns; internal gene duplication; intronization; spliceosomal introns
20.  Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns 
Nucleic Acids Research  2003;31(4):1121-1135.
As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5′ end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns’ features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts.
PMCID: PMC150231  PMID: 12582231
21.  Minor introns are embedded molecular switches regulated by highly unstable U6atac snRNA 
eLife  2013;2:e00780.
Eukaryotes have two types of spliceosomes, comprised of either major (U1, U2, U4, U5, U6) or minor (U11, U12, U4atac, U6atac; <1%) snRNPs. The high conservation of minor introns, typically one amidst many major introns in several hundred genes, despite their poor splicing, has been a long-standing enigma. Here, we discovered that the low abundance minor spliceosome’s catalytic snRNP, U6atac, is strikingly unstable (t½<2 hr). We show that U6atac level depends on both RNA polymerases II and III and can be rapidly increased by cell stress-activated kinase p38MAPK, which stabilizes it, enhancing mRNA expression of hundreds of minor intron-containing genes that are otherwise suppressed by limiting U6atac. Furthermore, p38MAPK-dependent U6atac modulation can control minor intron-containing tumor suppressor PTEN expression and cytokine production. We propose that minor introns are embedded molecular switches regulated by U6atac abundance, providing a novel post-transcriptional gene expression mechanism and a rationale for the minor spliceosome’s evolutionary conservation.
eLife digest
The central dogma of biology states that genetic material, DNA, is transcribed into RNA, which is then translated into proteins. However, the genes of many organisms have stretches of non-coding DNA that interrupt the sequences that code for protein. These non-coding sequences, which are called introns, must be removed, and the remaining sequences—which are called exons—must then be joined together to produce a messenger RNA (mRNA) transcript that is ready to be translated into protein.
The process of removing the introns and joining the exons is called splicing, and it is carried out by a molecular machine called the spliceosome. However, in addition to containing typical (‘major’) introns, several hundred human genes also contain a single ‘minor’ intron, and a minor spliceosome is needed to remove it. Minor introns occur in many highly conserved genes, but they are often inefficiently spliced. This means that the resulting mRNA transcripts may not be translated into proteins—which is puzzling given that these proteins perform important roles within the cell.
The major and minor spliceosomes are composed of proteins and small non-coding RNA molecules (which, as their name suggests, are never translated in cells). Now Younis et al. shed new light on the minor spliceosome by showing that a small non-coding RNA molecule known as U6atac, which catalyzes the removal of introns by the minor spliceosome, is highly unstable in human cells. This means that U6atac is a limiting factor for the splicing of minor introns—a process that is already limited by the very low abundance of the minor spliceosome under normal conditions. However, Younis et al. found that this bottleneck could be relieved by halting the degradation of U6atac. Experiments showed that U6atac can be stabilized by a key signaling molecule, a protein kinase (called p38MAPK), which is activated in response to stress. The resulting higher levels of U6atac promoted splicing of the introns in its target mRNA transcripts, and also modulated various signaling pathways in the cells.
Together, these results imply that the minor spliceosome is used as a valve that can help cells to adapt to stress and other changes. Moreover, by helping to translate mRNA transcripts that are already present in cells, it enables proteins to be produced rapidly in response to stress, bypassing the need for a fresh round of transcription.
PMCID: PMC3728624  PMID: 23908766
snRNA; U6atac; splicing; gene regulation; Human
22.  GenePainter: a fast tool for aligning gene structures of eukaryotic protein families, visualizing the alignments and mapping gene structures onto protein structures 
BMC Bioinformatics  2013;14:77.
All sequenced eukaryotic genomes have been shown to possess at least a few introns. This includes those unicellular organisms, which were previously suspected to be intron-less. Therefore, gene splicing must have been present at least in the last common ancestor of the eukaryotes. To explain the evolution of introns, basically two mutually exclusive concepts have been developed. The introns-early hypothesis says that already the very first protein-coding genes contained introns while the introns-late concept asserts that eukaryotic genes gained introns only after the emergence of the eukaryotic lineage. A very important aspect in this respect is the conservation of intron positions within homologous genes of different taxa.
GenePainter is a standalone application for mapping gene structure information onto protein multiple sequence alignments. Based on the multiple sequence alignments the gene structures are aligned down to single nucleotides. GenePainter accounts for variable lengths in exons and introns, respects split codons at intron junctions and is able to handle sequencing and assembly errors, which are possible reasons for frame-shifts in exons and gaps in genome assemblies. Thus, even gene structures of considerably divergent proteins can properly be compared, as it is needed in phylogenetic analyses. Conserved intron positions can also be mapped to user-provided protein structures. For their visualization GenePainter provides scripts for the molecular graphics system PyMol.
GenePainter is a tool to analyse gene structure conservation providing various visualization options. A stable version of GenePainter for all operating systems as well as documentation and example data are available at
PMCID: PMC3605371  PMID: 23496949
Exon; Intron; Gene structure; Evolution
23.  Phase distribution of spliceosomal introns: implications for intron origin 
The origin of spliceosomal introns is the central subject of the introns-early versus introns-late debate. The distribution of intron phases is non-uniform, with an excess of phase-0 introns. Introns-early explains this by speculating that a fraction of present-day introns were present between minigenes in the progenote and therefore must lie in phase-0. In contrast, introns-late predicts that the nonuniformity of intron phase distribution reflects the nonrandomness of intron insertions.
In this paper, we tested the two theories using analyses of intron phase distribution. We inferred the evolution of intron phase distribution from a dataset of 684 gene orthologs from seven eukaryotes using a maximum likelihood method. We also tested whether the observed intron phase distributions from 10 eukaryotes can be explained by intron insertions on a genome-wide scale. In contrast to the prediction of introns-early, the inferred evolution of intron phase distribution showed that the proportion of phase-0 introns increased over evolution. Consistent with introns-late, the observed intron phase distributions matched those predicted by an intron insertion model quite well.
Our results strongly support the introns-late hypothesis of the origin of spliceosomal introns.
PMCID: PMC1574350  PMID: 16959043
24.  Why eukaryotic cells use introns to enhance gene expression: Splicing reduces transcription-associated mutagenesis by inhibiting topoisomerase I cutting activity 
Biology Direct  2011;6:24.
The costs and benefits of spliceosomal introns in eukaryotes have not been established. One recognized effect of intron splicing is its known enhancement of gene expression. However, the mechanism regulating such splicing-mediated expression enhancement has not been defined. Previous studies have shown that intron splicing is a time-consuming process, indicating that splicing may not reduce the time required for transcription and processing of spliced pre-mRNA molecules; rather, it might facilitate the later rounds of transcription. Because the densities of active RNA polymerase II on most genes are less than one molecule per gene, direct interactions between the splicing apparatus and transcriptional complexes (from the later rounds of transcription) are infrequent, and thus unlikely to account for splicing-mediated gene expression enhancement.
Presentation of the hypothesis
The serine/arginine-rich protein SF2/ASF can inhibit the DNA topoisomerase I activity that removes negative supercoiling of DNA generated by transcription. Consequently, splicing could make genes more receptive to RNA polymerase II during the later rounds of transcription, and thus affect the frequency of gene transcription. Compared with the transcriptional enhancement mediated by strong promoters, intron-containing genes experience a lower frequency of cut-and-paste processes. The cleavage and religation activity of DNA strands by DNA topoisomerase I was recently shown to account for transcription-associated mutagenesis. Therefore, intron-mediated enhancement of gene expression could reduce transcription-associated genome instability.
Testing the hypothesis
Experimentally test whether transcription-associated mutagenesis is lower in intron-containing genes than in intronless genes. Use bioinformatic analysis to check whether exons flanking lost introns have higher frequencies of short deletions.
Implications of the hypothesis
The mechanism of intron-mediated enhancement proposed here may also explain the positive correlation observed between intron size and gene expression levels in unicellular organisms, and the greater number of intron containing genes in higher organisms.
This article was reviewed by Dr Arcady Mushegian, Dr Igor B Rogozin (nominated by Dr I King Jordan) and Dr Alexey S Kondrashov. For the full reviews, please go to the Reviewer's Reports section.
PMCID: PMC3118952  PMID: 21592350
25.  Phylogenetic Distribution of Intron Positions in Alpha-Amylase Genes of Bilateria Suggests Numerous Gains and Losses 
PLoS ONE  2011;6(5):e19673.
Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that “resets” of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures.
PMCID: PMC3096672  PMID: 21611157

Results 1-25 (768951)