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Analysis of intron gain and loss in fungal genomes provides support for an intron-rich fungus-animal ancestor.

Background

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.

Results

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.

Conclusion

Our results underscore the high intron density of eukaryotic ancestors and the widespread importance of intron loss through eukaryotic evolution.

Background

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.

Results

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.

Conclusion

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.

Reviewers

this article was reviewed by W. Ford Doolittle, James Darnell (nominated by W. Ford Doolittle), William Martin, and Anthony Poole.

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.

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.

Background

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.

Results

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.

Conclusion

Our results strongly support the introns-late hypothesis of the origin of spliceosomal introns.

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.

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.

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.

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.

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.

Synopsis

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.

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.

Results:

We developed a probabilistic model of evolution that allows for variability of intron gain and loss rates over branches of the phylogenetic tree, individual genes, and individual sites. Applying this model to an extended set of conserved eukaryotic genes, we find that parallel gain, on average, accounts for only ~8% of the shared intron positions. However, the distribution of parallel gains over the phylogenetic tree of eukaryotes is highly non-uniform. There are, practically, no parallel gains in closely related lineages, whereas for distant lineages, such as animals and plants, parallel gains appear to contribute up to 20% of the shared intron positions. In accord with these findings, we estimated that ancestral introns have a high probability to be retained in extant genomes, and conversely, that a substantial fraction of extant introns have retained their positions since the early stages of eukaryotic evolution. In addition, the density of sites that are available for intron insertion is estimated to be, approximately, one in seven basepairs.

Conclusion:

We obtained robust estimates of the contribution of parallel gain to the observed sharing of intron positions between eukaryotic species separated by different evolutionary distances. The results indicate that, although the contribution of parallel gains varies across the phylogenetic tree, the high level of intron position sharing is due, primarily, to evolutionary conservation. Accordingly, numerous introns appear to persist in the same position over hundreds of millions of years of evolution. This is compatible with recent observations of a negative correlation between the rate of intron gain and coding sequence evolution rate of a gene, suggesting that at least some of the introns are functionally relevant.

Evolutionary reconstructions using maximum likelihood methods point to unexpectedly high densities of introns in protein-coding genes of ancestral eukaryotic forms including the last common ancestor of all extant eukaryotes. Combined with the evidence of the origin of spliceosomal introns from invading Group II self-splicing introns, these results suggest that early ancestral eukaryotic genomes consisted of up to 80% sequences derived from Group II introns, a much greater contribution of introns than that seen in any extant genome. An organism with such an unusual genome architecture could survive only under conditions of a severe population bottleneck.

Background

Although large scale informatics studies on introns can be useful in making broad inferences concerning patterns of intron gain and loss, more specific questions about intron evolution at a finer scale can be addressed using a gene family where structure and function are well known. Genome wide surveys of tetraspanins from a broad array of organisms with fully sequenced genomes are an excellent means to understand specifics of intron evolution. Our approach incorporated several new fully sequenced genomes that cover the major lineages of the animal kingdom as well as plants, protists and fungi. The analysis of exon/intron gene structure in such an evolutionary broad set of genomes allowed us to identify ancestral intron structure in tetraspanins throughout the eukaryotic tree of life.

Methodology/Principal Findings

We performed a phylogenomic analysis of the intron/exon structure of the tetraspanin protein family. In addition, to the already characterized tetraspanin introns numbered 1 through 6 found in animals, three additional ancient, phase 0 introns we call 4a, 4b and 4c were found. These three novel introns in combination with the ancestral introns 1 to 6, define three basic tetraspanin gene structures which have been conserved throughout the animal kingdom. Our phylogenomic approach also allows the estimation of the time at which the introns of the 33 human tetraspanin paralogs appeared, which in many cases coincides with the concomitant acquisition of new introns. On the other hand, we observed that new introns (introns other than 1–6, 4a, b and c) were not randomly inserted into the tetraspanin gene structure. The region of tetraspanin genes corresponding to the small extracellular loop (SEL) accounts for only 10.5% of the total sequence length but had 46% of the new animal intron insertions.

Conclusions/Significance

Our results indicate that tests of intron evolution are strengthened by the phylogenomic approach with specific gene families like tetraspanins. These tests add to our understanding of genomic innovation coupled to major evolutionary divergence events, functional constraints and the timing of the appearance of evolutionary novelty.

Summary: Malin is a software package for the analysis of eukaryotic gene structure evolution. It provides a graphical user interface for various tasks commonly used to infer the evolution of exon–intron structure in protein-coding orthologs. Implemented tasks include the identification of conserved homologous intron sites in protein alignments, as well as the estimation of ancestral intron content, lineage-specific intron losses and gains. Estimates are computed either with parsimony, or with a probabilistic model that incorporates rate variation across lineages and intron sites.

Availability: Malin is available as a stand-alone Java application, as well as an application bundle for MacOS X, at the website http://www.iro.umontreal.ca/~csuros/introns/malin/. The software is distributed under a BSD-style license.

Contact: csuros@iro.umontreal.ca

Background

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.

Results

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.

Conclusions

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.

U12-type introns are spliced by the U12-dependent spliceosome and are present in the genomes of many higher eukaryotic lineages including plants, chordates and some invertebrates. However, due to their relatively recent discovery and a systematic bias against recognition of non-canonical splice sites in general, the introns defined by U12-type splice sites are under-represented in genome annotations. Such under-representation compounds the already difficult problem of determining gene structures. It also impedes attempts to study these introns genome-wide or phylum-wide. The resource described here, the U12 Intron Database (U12DB), aims to catalog the U12-type introns of completely sequenced eukaryotic genomes in a framework that groups orthologous introns with each other. This will aid further investigations into the evolution and mechanism of U12-dependent splicing as well as assist ongoing genome annotation efforts. Public access to the U12DB is available at .

The origin of present day introns is a subject of spirited debate. Any intron evolution theory must account for not only nuclear spliceosomal introns but also their antecedents. The evolution of group II introns is fundamental to this debate, since group II introns are the proposed progenitors of nuclear spliceosomal introns and are found in ancient genes from modern organisms. We have studied the evolution of chloroplast introns and twintrons (introns within introns) in the genus Euglena. Our hypothesis is that Euglena chloroplast introns arose late in the evolution of this lineage and that twintrons were formed by the insertion of one or more introns into existing introns. In the present study we find that 22 out of 26 introns surveyed in six different photosynthesis-related genes from the plastid DNA of Euglena gracilis are not present in one or more basally branching Euglena spp. These results are supportive of a late origin for Euglena chloroplast group II introns. The psbT gene in Euglena viridis, a basally branching Euglena species, contains a single intron in the identical position to a psbT twintron from E.gracilis, a derived species. The E.viridis intron, when compared with 99 other Euglena group II introns, is most similar to the external intron of the E.gracilis psbT twintron. Based on these data, the addition of introns to the ancestral psbT intron in the common ancester of E.viridis and E.gracilis gave rise to the psbT twintron in E.gracilis.

Images

Background

Spliceosomal introns are an ancient, widespread hallmark of eukaryotic genomes. Despite much research, many questions regarding the origin and evolution of spliceosomal introns remain unsolved, partly due to the difficulty of inferring ancestral gene structures. We circumvent this problem by using genes originated by endosymbiotic gene transfer, in which an intron-less structure at the time of the transfer can be assumed.

Results

By comparing the exon-intron structures of 64 mitochondrial-derived genes that were transferred to the nucleus at different evolutionary periods, we can trace the history of intron gains in different eukaryotic lineages. Our results show that the intron density of genes transferred relatively recently to the nuclear genome is similar to that of genes originated by more ancient transfers, indicating that gene structure can be rapidly shaped by intron gain after the integration of the gene into the genome and that this process is mainly determined by forces acting specifically on each lineage. We analyze 12 cases of mitochondrial-derived genes that have been transferred to the nucleus independently in more than one lineage.

Conclusions

Remarkably, the proportion of shared intron positions that were gained independently in homologous genes is similar to that proportion observed in genes that were transferred prior to the speciation event and whose shared intron positions might be due to vertical inheritance. A particular case of parallel intron gain in the nad7 gene is discussed in more detail.

Abstract

Continued improvements in Next-Generation DNA/RNA sequencing coupled with advances in gene annotation have provided researchers access to a plethora of annotated genomes. Subsequent analyses of orthologous gene structures have identified numerous intron gain and loss events that have occurred both recently and in the very distant past. This research has afforded exceptional insight into the temporal and lineage-specific rates of intron gain and loss among various species throughout evolution. Numerous studies have also attempted to identify the molecular mechanisms of intron gain and loss. However, even after considerable effort, very little is known about these processes. In particular, the mechanism(s) of intron gain have proven exceptionally enigmatic and remain topics of considerable debate. Currently, there exists no definitive consensus as to what mechanism(s) may generate introns. Because many introns are known to affect gene expression, it is necessary to understand the molecular process(es) by which introns may be gained. Here we review the seven most commonly purported mechanisms of intron gain and, when possible, summarize molecular evidence for or against the occurrence of each of these mechanisms. Furthermore, we catalogue indirect evidence that supports the occurrence of each mechanism. Finally, because these proposed mechanisms fail to explain the mechanistic origin of many recently gained introns, we also look at trends that may aid researchers in identifying other potential mechanism(s) of intron gain.

Reviewers

This article was reviewed by Eugene Koonin, Scott Roy (nominated by W. Ford Doolittle), and John Logsdon.

Background

Spliceosomal introns are important components of eukaryotic genes as their structure, sizes and contents reflect the architecture of gene and genomes. Intron size, determined by both neutral evolution, repetitive elements activities and potential functional constraints, varies significantly in eukaryotes, suggesting unique dynamics and evolution in different lineages of eukaryotic organisms. However, the evolution of intron size, is rarely studied. To investigate intron size dynamics in flowering plants, in particular domesticated grapevines, a survey of intron size and content in wine grape (Vitis vinifera Pinot Noir) genes was conducted by assembling and mapping the transcriptome of V. vinifera genes from ESTs to characterize and analyze spliceosomal introns.

Results

Uncommonly large size of spliceosomal intron was observed in V. vinifera genome, otherwise inconsistent with overall genome size dynamics when comparing Arabidopsis, Populus and Vitis. In domesticated grapevine, intron size is generally not related to gene function. The composition of enlarged introns in grapevines indicated extensive transposable element (TE) activity within intronic regions. TEs comprise about 80% of the expanded intron space and in particular, recent LTR retrotransposon insertions are enriched in these intronic regions, suggesting an intron size expansion in the lineage leading to domesticated grapevine, instead of size contractions in Arabidopsis and Populus. Comparative analysis of selected intronic regions in V. vinifera cultivars and wild grapevine species revealed that accelerated TE activity was associated with grapevine domestication, and in some cases with the development of specific cultivars.

Conclusions

In this study, we showed intron size expansion driven by TE activities in domesticated grapevines, likely a result of long-term vegetative propagation and intensive human care, which simultaneously promote TE proliferation and repress TE removal mechanisms such as recombination. The intron size expansion observed in domesticated grapevines provided an example of rapid plant genome evolution in response to artificial selection and propagation, and may shed light on the important genomic changes during domestication. In addition, the transcriptome approach used to gather intron size data significantly improved annotations of the V. vinifera 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.

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.

Background

The origin and importance of exon-intron architecture comprises one of the remaining mysteries of gene evolution. Several studies have investigated the variations of intron length, GC content, ordinal position in a gene and divergence. However, there is little study about the structural variation of exons and introns.

Results

We investigated the length, GC content, ordinal position and divergence in both exons and introns of 13 eukaryotic genomes, representing plant and animal. Our analyses revealed that three basic patterns of exon-intron variation were present in nearly all analyzed genomes (P < 0.001 in most cases): an ordinal reduction of length and divergence in both exon and intron, a co-variation between exon and its flanking introns in their length, GC content and divergence, and a decrease of average exon (or intron) length, GC content and divergence as the total exon numbers of a gene increased. In addition, we observed that the shorter introns had either low or high GC content, and the GC content of long introns was intermediate.

Conclusion

Although the factors contributing to these patterns have not been identified, our results provide three important clues: common factor(s) exist and may shape both exons and introns; the ordinal reduction patterns may reflect a time-orderly evolution; and the larger first and last exons may be splicing-required. These clues provide a framework for elucidating mechanisms involved in the organization of eukaryotic genomes and particularly in building exon-intron structures.

Specific questions about intron evolution are precisely addressed applying a phylogenomic approach to suitable gene families. With this approach we have recently reported that the appearance of most human tetraspanins occurred in the common ancestor of vertebrates and coincides in nearly all cases with the concomitant acquisition of new introns. We observed that indels at the ends of the DNA exonic sequences with no involvement of the corresponding intronic sequence, were the cause of two discordant intron positions between orthologous tetraspanins. Here, we discuss a putative intron sliding occurrence in which a new acquired intron junction (intron 1a) in the ancestor of chordates could have been shifted to new positions (introns 1b and 1c) during the expansion of the tetraspanin family in vertebrates. Such a mechanism could be responsible for generating some of the variation of function in this important family of membrane spanning proteins.

Group II introns are ribozymes, removing themselves from their primary transcripts, as well as mobile genetic elements, transposing via an RNA intermediate, and are thought to be the ancestors of spliceosomal introns. Although common in bacteria and most eukaryotic organelles, they have never been reported in any bilaterian animal genome, organellar or nuclear. Here we report the first group II intron found in the mitochondrial genome of a bilaterian worm. This location is especially surprising, since animal mitochondrial genomes are generally distinct from those of plants, fungi, and protists by being small and compact, and so are viewed as being highly streamlined, perhaps as a result of strong selective pressures for fast replication while establishing germ plasm during early development. This intron is found in the mtDNA of an annelid worm, (an undescribed species of Nephtys), where the complete sequence revealed a 1819 bp group II intron inside the cox1 gene. We infer that this intron is the result of a recent horizontal gene transfer event from a viral or bacterial vector into the mitochondrial genome of Nephtys sp. Our findings hold implications for understanding mechanisms, constraints, and selective pressures that account for patterns of animal mitochondrial genome evolution

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.