The evolutionary gain of spliceosomal introns: sequence and phase preferences

Theories regarding the evolution of spliceosomal introns differ in the extent to which the distribution of introns reflects either a formative role in the evolution of protein-coding genes or the adventitious gain of genetic elements. Here, systematic methods are used to assess the causes of the present-day distribution of introns in 10 families of eukaryotic protein-coding genes comprising 1,868 introns in 488 distinct alignment positions. The history of intron evolution inferred using a probabilistic model that allows ancestral inheritance of introns, gain of introns, and loss of introns reveals that the vast majority of introns in these eukaryotic gene families were not inherited from the most recent common ancestral genes, but were gained subsequently. Furthermore, among inferred events of intron gain that meet strict criteria of reliability, the distribution of sites of gain with respect to reading-frame phase shows a 5:3:2 ratio of phases 0, 1 and 2, respectively, and exhibits a nucleotide preference for MAG GT (positions -3 to +2 relative to the site of gain). The nucleotide preferences of intron gain may prove to be the ultimate cause for the phase bias. The phase bias of intron gain is sufficient to account quantitatively for the well-known 5:3:2 bias in phase frequencies among extant introns, a conclusion that holds even when taxonomic heterogeneity in phase patterns is considered. Thus, intron gain accounts for the vast majority of extant introns and for the bias toward phase 0 introns that previously was interpreted as evidence for ancient formative introns.     

Phylogenetic distribution of intron positions in alpha-amylase genes of bilateria suggests numerous gains and losses

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.     

Widespread intron loss suggests retrotransposon activity in ancient apicomplexans

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

Formation of new genes explains lower intron density in mammalian Rhodopsin G protein-coupled receptors

Mammalian G protein-coupled receptor (GPCR) genes are characterised by a large proportion of intronless genes or a lower density of introns when compared with GPCRs of invertebrates. It is unclear which mechanisms have influenced intron density in this protein family, which is one of the largest in the mammalian genomes. We used a combination of Hidden Markov Models (HMM) and BLAST searches to establish the comprehensive repertoire of Rhodopsin GPCRs from seven species and performed overall alignments and phylogenetic analysis using the maximum parsimony method for over 1400 receptors in 12 subgroups. We identified 14 different Ancestral Receptor Groups (ARGs) that have members in both vertebrate and invertebrate species. We found that there exists a remarkable difference in the intron density among ancestral and new Rhodopsin GPCRs. The intron density among ARGs members was more than 3.5-fold higher than that within non-ARG members and more than 2-fold higher when considering only the 7TM region. This suggests that the new GPCR genes have been predominantly formed intronless while the ancestral receptors likely accumulated introns during their evolution. Many of the intron positions found in mammalian ARG receptor sequences were found to be present in orthologue invertebrate receptors suggesting that these intron positions are ancient. This analysis also revealed that one intron position is much more frequent than any other position and it is common for a number of phylogenetically different Rhodopsin GPCR groups. This intron position lies within a functionally important, conserved, DRY motif which may form a proto-splice site that could contribute to positional intron insertion. Moreover, we have found that other receptor motifs, similar to DRY, also contain introns between the second and third nucleotide of the arginine codon which also forms a proto-splice site. Our analysis presents compelling evidence that there was not a major loss of introns in mammalian GPCRs and formation of new GPCRs among mammals explains why these have fewer introns compared to invertebrate GPCRs. We also discuss and speculate about the possible role of different RNA- and DNA-based mechanisms of intron insertion and loss.     

Investigation of loss and gain of introns in the compact genomes of pufferfishes (Fugu and Tetraodon)

We have investigated intron evolution in the compact genomes of 2 closely related species of pufferfishes, Fugu rubripes and Tetraodon nigroviridis, that diverged about 32 million years ago (MYA). Analysis of 148,028 aligned intron positions in 13,547 gene pairs using human as an outgroup identified 57 and 24 intron losses in Tetraodon and fugu lineages, respectively, and no gain in either lineage. For comparison, we analyzed 144,545 intron positions in 12,866 orthologous pairs of genes in human and mouse that diverged about 61 MYA using dog as an outgroup and identified 51 intron losses in mouse and 3 losses in human and no gain. The rate of intron loss in Tetraodon is higher than that in fugu, mouse, and human but lower than the previous estimates for other eukaryotes. The introns lost in pufferfishes and mammals are significantly shorter than the mean size of introns in the genome. One intron deleted in fugu and another in Tetraodon have left behind 6 and 3 nucleotides, respectively, suggesting that they were lost due to genomic deletions. Such losses of introns are likely to be the result of a higher rate of DNA deletions experienced by the genomes of pufferfishes compared with mammals. The shorter generation time of Tetraodon compared with fugu, and the rich diversity and higher activity of transposable elements in pufferfishes compared with mammals, may be responsible for the higher rate of intron loss in Tetraodon. Our findings indicate that overall very little intron turnover has occurred in pufferfishes and mammals during recent evolution and that intron gain is an extremely rare event in vertebrate evolution.     

Novel Introner-Like Elements in fungi Are Involved in Parallel Gains of Spliceosomal Introns

Spliceosomal introns are key components of the eukaryotic gene structure. Although they contributed to the emergence of eukaryotes, their origin remains elusive. In fungi, they might originate from the multiplication of invasive introns named Introner-Like Elements (ILEs). However, so far ILEs have been observed in six fungal species only, including Fulvia fulva and Dothistroma septosporum (Dothideomycetes), arguing against ILE insertion as a general mechanism for intron gain. Here, we identified novel ILEs in eight additional fungal species that are phylogenetically related to F. fulva and D. septosporum using PCR amplification with primers derived from previously identified ILEs. The ILE content appeared unique to each species, suggesting independent multiplication events. Interestingly, we identified four genes each containing two gained ILEs. By analysing intron positions in orthologues of these four genes in Ascomycota, we found that three ILEs had inserted within a 15 bp window that contains regular spliceosomal introns in other fungal species. These three positions are not the result of intron sliding because ILEs are newly gained introns. Furthermore, the alternative hypothesis of an inferred ancestral gain followed by independent losses contradicts the observed degeneration of ILEs. These observations clearly indicate three parallel intron gains in four genes that were randomly identified. Our findings suggest that parallel intron gain is a phenomenon that has been highly underestimated in ILE-containing fungi, and likely in the whole fungal kingdom.     

Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns

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.     

Intron gain and loss in the evolution of the conserved eukaryotic recombination machinery

Intron conservation, intron gain or loss and putative intron sliding events were determined for a set of three genes (SPO11, MRE11 and DMC1) involved in basic aspects of recombination in eukaryotes. These are ancient genes and present in nearly all of the major kingdoms. MRE11 is of bacterial origin and can be found in all kingdoms. DMC1 is a specialized homolog of the bacterial RecA protein, whereas the SPO11 gene is of archaebacterial origin. Only unique homologs of SPO11 are found in animals and fungi whereas three distantly related SPO11 copies are present in plant genomes. A comparison of the respective intron positions and phases of all genes was performed, demonstrating that a quarter of the intron positions were perfectly conserved over more than 1000000000 years. Regarding the remaining three quarters of the introns we found insertions to be about three times more frequent than deletions. Aligning the introns of the three different SPO11 homologs of Arabidopsis thaliana we propose a conclusive model of their evolution. We postulate that at least one duplication event occurred shortly after the divergence of plants from animals and fungi and that a respective homolog has been retained in a protist group, the apicomplexa.     

Analysis of evolution of exon-intron structure of eukaryotic genes

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

Conservation of the positions of metazoan introns from sponges to humans

Sponges (phylum Porifera) are the phylogenetic oldest Metazoa still extant. They can be considered as reference animals (Urmetazoa) for the understanding of the evolutionary processes resulting in the creation of Metazoa in general and also for the metazoan gene organization in particular. In the marine sponge Suberites domuncula, genes encoding p38 and JNK kinases contain nine and twelve introns, respectively. Eight introns in both genes share the same positions and the identical phases. One p38 intron slipped for six bases and the JNK gene has three more introns. However, the sequences of the introns are not conserved and the introns in JNK gene are generally much longer. Introns interrupt most of the conserved kinase subdomains I-XI and are found in all three phases (0, 1 and 2). We analyzed in details p38 and JNK genes from human, Caenorhabditis elegans and Drosophila melanogaster and found in most genes introns at the positions identical to those in sponge genes. The exceptions are two p38 genes from D. melanogaster that have lost all introns in the coding sequence. The positions of 11 introns in each of four human p38 genes are fully conserved and ten introns occupy identical positions as the introns in sponge p38 or JNK genes. The same is true for nine, out of ten introns in the human JNK-1 gene. The introns in human p38 and JNK genes are on average more than ten times longer than corresponding introns in sponges. It was proposed that yeast HOG1-like kinases (from i.e. Saccharomyces cerevisiae and Emericella nidulans) and metazoan p38 and JNK kinases are orthologues. p38 and JNK genes were created after the split from fungi by the duplication and diversification of the HOG1-like progenitor gene. Our results further support the common origin of p38 and JNK genes and speak in favor of a very early time of duplication. The ancestral gene contained at least ten introns, which are still present at the very conserved positions in p38 and JNK genes of extant animals. Four of these introns are present at the same positions in the HOG-like gene in the fungus E. nidulans. The others probably entered the ancestral gene after the split of fungi, but before the duplication of the gene and before the creation of the common, urmetazoan progenitor of all multicellular animals. A second gene coding for an immune molecule is described, the allograft inflammatory factor, which likewise showed a highly conserved exon/intron structure in S. domuncula and in human. These data show that the intron/exon borders are highly conserved in genes from sponges to human.     

Complex Evolutionary Patterns of tRNAUAALeu Group I Introns in Cyanobacterial Radiation

Based on the findings that plastids and cyanobacteria have similar group I introns inserted into tRNAUAALeu genes, these introns have been suggested to be immobile and of ancient origin. In contrast, recent evidence suggests lateral transfer of cyanobacterial group I introns located in tRNAUAALeu genes. In light of these new findings, we have readdressed the evolution and lateral transfer of tRNAUAALeu group I introns in cyanobacteral radiation. We determined the presence of introns in 38 different strains, representing the major cyanobacterial lineages, and characterized the introns in 22 of the strains. Notably, two of these strains have two tRNAUAALeu genes, with each of these genes interrupted by introns, while three of the strains have both interrupted and uninterrupted genes. Two evolutionary distinct clusters of tRNA genes, with the genes interrupted by introns belonging to two distinct intron clusters, were identified. We also compared 16S rDNA and intron evolution for both closely and distantly related strains. The distribution of the introns in the clustered groups, as defined from 16S rDNA analysis, indicates relatively recent gain and/or loss of the introns in some of these lineages. The comparative analysis also suggests differences in the phylogenetic trees for 16S rDNA and the tRNAUAALeu group I introns. Taken together, our results show that the evolution of the intron is considerably more complex than previous studies found to be the case. We discuss, based on our results, evolutionary models involving lateral intron transfer and models involving differential loss of the intron.     

Vestiges of lost introns in the thermal stability map of DNA

The absence of introns in prokaryotic genes has been explained by intron loss on various bases. Here we report another piece of evidence on intron loss, which was found in the thermal stability map of DNA. We calculated the local melting temperature of cDNA sequences and found that (i) gaps in thermal stability tend to occur near intron positions with a statistical significance, and (ii) one-third of the gaps far from intron positions can be assigned to lost introns. From these results we conclude that the gaps of thermal stability in protein coding regions are the vestiges of lost introns.     

Generation of a database containing discordant intron positions in eukaryotic genes (MIDB)

MOTIVATION: Intron sliding is the relocation of intron-exon boundaries over short distances and is often also referred to as intron slippage or intron migration or intron drift. We have generated a database containing discordant intron positions in homologous genes (MIDB--Mismatched Intron DataBase). Discordant intron positions are those that are either closely located in homologous genes (within a window of 10 nucleotides) or an intron position that is present in one gene but not in any of its homologs. The MIDB database aims at systematically collecting information about mismatched introns in the genes from GenBank and organizing it into a form useful for understanding the genomics and dynamics of introns thereby helping understand the evolution of genes. RESULTS: Intron displacement or sliding is critically important for explaining the present distribution of introns among orthologous and paralogous genes. MIDB allows examining of intron movements and allows mapping of intron positions from homologous proteins onto a single sequence. The database is of potential use for molecular biologists in general and for researchers who are interested in gene evolution and eukaryotic gene structure. Partial analysis of this database allowed us to identify a few putative cases of intron sliding. AVAILABILITY: http://intron.bic.nus.edu.sg/midb/midb.html     

Tempo and Mode of Spliceosomal Intron Evolution in Actin of Foraminifera

Spliceosomal introns are present in almost all eukaryotic genes, yet little is known about their origin and turnover in the majority of eukaryotic phyla. There is no agreement whether most introns are ancestral and have been lost in some lineage or have been gained recently. We addressed this question by analyzing the spatial and temporal distribution of introns in actins of foraminifera, a group of testate protists whose exceptionally rich fossil record permits the calibration of molecular phylogenies to date intron origins. We identified 24 introns dispersed along the sequence of two foraminiferan actin paralogues and actin deviating proteins, an unconventional type of fast-evolving actin found in some foraminifera. Comparison of intron positions indicates that 20 of 24 introns are specific to foraminifera. Four introns shared between foraminifera and other eukaryotes were interpreted as parallel gains because they have been found only in single species belonging to phylogenetically distinctive lineages. Moreover, additional recent intron gain due to the transfer between the actin paralogues was observed in two cultured species. Based on a relaxed molecular clock timescale, we conclude that intron gains in actin took place throughout the evolution of foraminifera, with the oldest introns inserted between 550 and 500 million years ago and the youngest ones acquired less than 100 million years ago. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Debashish Bhattacharya]     

Evolution and structure of the fibrinogen genes: Random insertion of introns or selective loss?

Chromosomal linkage as well as sequence homologies provide unequivocal evidence that the genes for the alpha, beta and gamma chains of fibrinogen arose by successive duplication of a single ancestral gene. Yet, when the three fibrinogen chains are aligned by amino acid homology, the positions of intervening sequences coincide at only two positions for all three chains. While one additional intron occurs at a homologous site in the beta and gamma chains, none of the positions of the remaining 11 introns in the three genes is shared. This arrangement of introns in the three fibrinogen genes suggests that either introns were selectively lost, implying that there is essential information in the retained introns, or the common introns were present in the ancestral fibrinogen gene and introns have been randomly inserted since the triplication of the original gene. The more likely possibility of selective loss of introns implies that the ancestral gene, as it existed about one billion years ago, must have been composed of numerous small exons.     

Higher intron loss rate in Arabidopsis thaliana than A. lyrata is consistent with stronger selection for a smaller genome

The number of introns varies considerably among different organisms. This can be explained by the differences in the rates of intron gain and loss. Two factors that are likely to influence these rates are selection for or against introns and the mutation rate that generates the novel intron or the intronless copy. Although it has been speculated that stronger selection for a compact genome might result in a higher rate of intron loss and a lower rate of intron gain, clear evidence is lacking, and the role of selection in determining these rates has not been established. Here, we studied the gain and loss of introns in the two closely related species Arabidopsis thaliana and A. lyrata as it was recently shown that A. thaliana has been undergoing a faster genome reduction driven by selection. We found that A. thaliana has lost six times more introns than A. lyrata since the divergence of the two species but gained very few introns. We suggest that stronger selection for genome reduction probably resulted in the much higher intron loss rate in A. thaliana, although further analysis is required as we could not find evidence that the loss rate increased in A. thaliana as opposed to having decreased in A. lyrata compared with the rate in the common ancestor. We also examined the pattern of the intron gains and losses to better understand the mechanisms by which they occur. Microsimilarity was detected between the splice sites of several gained and lost introns, suggesting that nonhomologous end joining repair of double-strand breaks might be a common pathway not only for intron gain but also for intron loss.