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.     

Conservation versus parallel gains in intron evolution

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

Evidence for extensive recent intron transposition in closely related fungi

Though spliceosomal introns are a major structural component of most eukaryotic genes and intron density varies by more than three orders of magnitude among eukaryotes [1-3], the origins of introns are poorly understood, and only a few cases of unambiguous intron gain are known [4-8]. We utilized population genomic comparisons of three closely related fungi to identify crucial transitory phases of intron gain and loss. We found 74 intron positions showing intraspecific presence-absence polymorphisms (PAPs) for the entire intron. Population genetic analyses identified intron PAPs at different stages of fixation and showed that intron gain or loss was very recent. We found direct support for extensive intron transposition among unrelated genes. A substantial proportion of highly similar introns in the genome either were recently gained or showed a transient phase of intron PAP. We also identified an intron transfer among paralogous genes that created a new intron. Intron loss was due mainly to homologous recombination involving reverse-transcribed mRNA. The large number of intron positions in transient phases of either intron gain or loss shows that intron evolution is much faster than previously thought and provides an excellent model to study molecular mechanisms of intron gain.     

An analysis of intron positions in relation to nucleotides, amino acids, and protein secondary structure

We present an analysis of intron positions in relation to nucleotides, amino acid residues, and protein secondary structure. Previous work has shown that intron sites in proteins are not randomly distributed with respect to secondary structures. Here we show that this preference can be almost totally explained by the nucleotide bias of splice site machinery, and may well not relate to protein stability or conformation at all. Each intron phase is preferentially associated with its own set of residues: phase 0 introns with lysine, glutamine, and glutamic acid before the intron, and valine after; phase 1 introns with glycine, alanine, valine, aspartic acid, and glutamic acid; and phase 2 introns with arginine, serine, lysine, and tryptophan. These preferences can be explained principally on the basis of nucleotide bias at intron locations, which is in accordance with previous literature. Although this work does not prove that introns are inserted into genomes at specific proto-splice sites, it shows that the nucleotide bias surrounding introns, however it originally occurred, explains the observed correlations between introns and protein secondary structure.     

The Agaricus bisporus cox1 gene: the longest mitochondrial gene and the largest reservoir of mitochondrial group i introns

In eukaryotes, introns are located in nuclear and organelle genes from several kingdoms. Large introns (up to 5 kbp) are frequent in mitochondrial genomes of plant and fungi but scarce in Metazoa, even if these organisms are grouped with fungi among the Opisthokonts. Mitochondrial introns are classified in two groups (I and II) according to their RNA secondary structure involved in the intron self-splicing mechanism. Most of these mitochondrial group I introns carry a "Homing Endonuclease Gene" (heg) encoding a DNA endonuclease acting in transfer and site-specific integration ("homing") and allowing intron spreading and gain after lateral transfer even between species from different kingdoms. Opposed to this gain mechanism, is another which implies that introns, which would have been abundant in the ancestral genes, would mainly evolve by loss. The importance of both mechanisms (loss and gain) is matter of debate. Here we report the sequence of the cox1 gene of the button mushroom Agaricus bisporus, the most widely cultivated mushroom in the world. This gene is both the longest mitochondrial gene (29,902 nt) and the largest group I intron reservoir reported to date with 18 group I and 1 group II. An exhaustive analysis of the group I introns available in cox1 genes shows that they are mobile genetic elements whose numerous events of loss and gain by lateral transfer combine to explain their wide and patchy distribution extending over several kingdoms. An overview of intron distribution, together with the high frequency of eroded heg, suggests that they are evolving towards loss. In this landscape of eroded and lost intron sequences, the A. bisporus cox1 gene exhibits a peculiar dynamics of intron keeping and catching, leading to the largest collection of mitochondrial group I introns reported to date in a Eukaryote.     

USING RDNA GENES TO UNDERSTAND THE ORIGIN AND EVOLUTION OF SPLICEOSOMAL AND GROUP I INTRONS

Ribosomal DNA genes in lichen algae and lichen fungi are astonishingly rich in spliceosomal and group I introns. We use phylogenetic, secondary structure, and biochemical analyses to understand the evolution of these introns. Despite the widespread distribution of spliceosomal introns in nuclear pre-mRNA genes, their general mechanism of origin remains an open question because few proven cases of recent and pervasive intron origin have been documented. The lichen introns are valuable in this respect because they are undoubtedly of a "recent" origin and limited to the Euascomycetes. Our analyses suggest that rDNA spliceosomal introns have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into r RNAs. We propose that the spliceosome itself (and not an external agent; e.g. transposable elements, group II introns) has given rise to the introns. The rDNA introns are found most often between the flanking sequence G (78%) - intron-G (72%), and their clustered positions on secondary structures suggest that particular r RNA regions are preferred sites (i.e., proto-splice sites) for insertion. Mapping of intron positions on the newly available tertiary structures show that they are found most often in exposed regions of the ribosomes. This again is consistent with an intron origin through reverse-splicing. Remarkably, the distribution and phylogenetic relationships of most group I introns in nuclear rDNA genes are also consistent with a reverse-splicing origin. These data underline the value of lichens as a model system for understanding intron origin and stress the importance of RNA-level processes in the spread of these sequences in nuclear coding regions.     

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.     

Testing the "proto-splice sites" model of intron origin: evidence from analysis of intron phase correlations

A few nucleotide sites of nuclear exons that flank introns are often conserved. A hypothesis has suggested that these sites, called "proto-splice sites," are remnants of recognition signals for the insertion of introns in the early evolution of eukaryotic genes. This notion of proto-splice sites has been an important basis for the insertional theory of introns. This hypothesis predicts that the distribution of proto-splice sites would determine the distribution of intron phases, because the positions of introns are just a subset of the proto-splice sites. We previously tested this prediction by examining the proportions of the phases of proto-splice sites, revealing nothing in these proportion distributions similar to observed proportions of intron phases. Here, we provide a second independent test of the proto-splice site hypothesis, with regard to its prediction that the proto-splice sites would mimic intron phase correlations, using a CDS database we created from GenBank. We tested four hypothetical proto-splice sites G / G, AG / G, AG / GT, and C/AAG / R. Interestingly, while G / G and AG / GT site phase distributions are not consistent with actual introns, we observed that AG / G and C/AAG / R sites have a symmetric phase excess. However, the patterns of the excess are quite different from the actual intron phase distribution. In addition, particular amino acid repeats in proteins were found to partially contribute to the excess of symmetry at these two types of sites. The phase associations of all four sites are significantly different from those of intron phases. Furthermore, a general model of intron insertion into proto-splice sites was simulated by Monte Carlo simulation to investigate the probability that the random insertion of introns into AG / G and C/AAG / R sites could generate the observed intron phase distribution. The simulation showed that (1) no observed correlation of intron phases was statistically consistent with the phase distribution of proto-splice sites in the simulated virtual genes; (2) most conservatively, no simulation in 10,000 Monte Carlo experiments gave a pattern with an excess of symmetric (1, 1) exons larger than those of (0, 0) and (2, 2), a major statistical feature of intron phase distribution that is consistent with the directly observed cases of exon shuffling. Thus, these results reject the null hypothesis that introns are randomly inserted into preexisting proto-splice sites, as suggested by the insertional theory of introns.     

Three variant introns of the same general class in the mitochondrial gene for cytochrome oxidase subunit 1 in Aspergillus nidulans

The oxiA gene of Aspergillus nidulans, coding for cytochrome oxidase subunit 1, is shown by DNA sequencing to contain three introns. An AUG start codon is not present at the beginning of the sequence, suggesting that either another codon, possibly the four base codon AUGA, is used for initiation or there is a further short intron between the true start codon and the beginning of the recognisable coding region. The second and third introns have long open reading frames, which could code for maturase proteins. The lack of conservation of amino acid sequence in the putative region of proteolytic cleavage for maturase formation suggests that the first conserved decapeptide may act as the recognition signal for protein processing. The third intron is remarkably (70%) homologous to the second intron of the cytochrome oxidase subunit 1 gene of Schizosaccharomyces pombe and both are located in exactly the same position. The third Aspergillus intron has an in-frame insertion of a 37-bp GC-rich DNA sequence which is now flanked by a 5-bp repeat, a well-known feature of transposable elements. All three introns in the oxiA gene have a 'core' RNA secondary structure found in a class of introns fitting the RNA splicing model of Davies et al. (1982). This core RNA structure may play a catalytic as well as a structural role in intron splicing. A sequence within the intron could act as a guide to align the splice sites of two of the introns in accordance with the model of Davies et al.     

High rate of recent intron gain and loss in simultaneously duplicated Arabidopsis genes

We examined the gene structure of a set of 2563 Arabidopsis thaliana paralogous pairs that were duplicated simultaneously 20-60 MYA by tetraploidy. Out of a total of 23,164 introns in these genes, we found that 10,004 pairs have been conserved and 578 introns have been inserted or deleted in the time since the duplication event. This intron insertion/deletion rate of 2.7 x 10(-3) to 9.1 x 10(-4) per site per million years is high in comparison to previous studies. At least 56 introns were gained and 39 lost based on parsimony analysis of the phylogenetic distribution of these introns. We found weak evidence that genes undergoing intron gain and loss are biased with respect to gene ontology terms. Gene pairs that experienced at least 2 intron insertions or deletions show evidence of enrichment for membrane location and transport and transporter activity function. We do not find any relationship of intron flux to expression level or G + C content of the gene. Detection of a bias in the location of intron gains and losses within a gene depends on the method of measurement: an intragene method indicates that events (specifically intron losses) are biased toward the 3' end of the gene. Despite the relatively recent acquisition of these introns, we found only one case where we could identify the mechanism of intron origin--the TOUCH3 gene has experienced 2 tandem, partial, internal gene duplications that duplicated a preexisting intron and also created a novel, alternatively spliced intron that makes use of a duplicated pair of cryptic splice sites.     

A general model for the evolution of nuclear pre-mRNA introns

We present an overview of the evolution of eukaryotic split gene structure and pre-mRNA splicing mechanisms. We have drawn together several seemingly conflicting ideas and we show that they can all be incorporated in a single unified theory of intron evolution. The resulting model is consistent with the notion that introns, as a class, are very ancient, having originated in the "RNA world"; it also supports the concept that introns may have played a crucial role in the construction of many eukaryotic genes and it accommodates the idea that introns are related to mobile insertion elements. Our conclusion is that introns could have a profound effect on the course of eukaryotic gene evolution, but that the origin and maintenance of intron sequences depends, largely, on natural selection acting on the intron sequences themselves.     

Evidence for Transitional Stages in the Evolution of Euglenid Group II Introns and Twintrons in the Monomorphina aenigmatica Plastid Genome

Background

Photosynthetic euglenids acquired their plastid by secondary endosymbiosis of a prasinophyte-like green alga. But unlike its prasinophyte counterparts, the plastid genome of the euglenid Euglena gracilis is riddled with introns that interrupt almost every protein-encoding gene. The atypical group II introns and twintrons (introns-within-introns) found in the E. gracilis plastid have been hypothesized to have been acquired late in the evolution of euglenids, implying that massive numbers of introns may be lacking in other taxa. This late emergence was recently corroborated by the plastid genome sequences of the two basal euglenids, Eutreptiella gymnastica and Eutreptia viridis, which were found to contain fewer introns.

Methodology/Principal Findings

To gain further insights into the proliferation of introns in euglenid plastids, we have characterized the complete plastid genome sequence of Monomorphina aenigmatica, a freshwater species occupying an intermediate phylogenetic position between early and late branching euglenids. The M. aenigmatica UTEX 1284 plastid genome (74,746 bp, 70.6% A+T, 87 genes) contains 53 intron insertion sites, of which 41 were found to be shared with other euglenids including 12 of the 15 twintron insertion sites reported in E. gracilis.

Conclusions

The pattern of insertion sites suggests an ongoing but uneven process of intron gain in the lineage, with perhaps a minimum of two bursts of rapid intron proliferation. We also identified several sites that represent intermediates in the process of twintron evolution, where the external intron is in place, but not the internal one, offering a glimpse into how these convoluted molecular contraptions originate.     

Splicing-Related Features of Introns Serve to Propel Evolution

The role of spliceosomal intronic structures played in evolution has only begun to be elucidated. Comparative genomic analyses of fungal snoRNA sequences, which are often contained within introns and/or exons, revealed that about one-third of snoRNA-associated introns in three major snoRNA gene clusters manifested polymorphisms, likely resulting from intron loss and gain events during fungi evolution. Genomic deletions can clearly be observed as one mechanism underlying intron and exon loss, as well as generation of complex introns where several introns lie in juxtaposition without intercalating exons. Strikingly, by tracking conserved snoRNAs in introns, we found that some introns had moved from one position to another by excision from donor sites and insertion into target sties elsewhere in the genome without needing transposon structures. This study revealed the origin of many newly gained introns. Moreover, our analyses suggested that intron-containing sequences were more prone to sustainable structural changes than DNA sequences without introns due to intron''s ability to jump within the genome via unknown mechanisms. We propose that splicing-related structural features of introns serve as an additional motor to propel evolution.     

Nucleotide sequence and intron structure of the apocytochrome b gene of Neurospora crassa mitochondria

The sequence of the apocytochrome b (cob) gene of Neurospora crassa has been determined. The structural gene is interrupted by two intervening sequences of approximately 1260 bp each. The polypeptide encoded by the exons shows extensive homology with the cob proteins of Aspergillus nidulans and Saccharomyces cerevisiae (79% and 60%, respectively). The two introns are, however, located at sites different from those of introns in the cob genes of A. nidulans and S. cerevisiae (which contain highly homologous introns at the same site within the gene). The introns share several short regions of sequence homology (10-12 bp long) with each other and with other fungal mitochondrial introns. Moreover, the second intron contains a 50 nucleotide long sequence that is highly homologous with sequences within every ribosomal intron of fungal mitochondria sequenced to date. The conserved sequences may allow the formation of a core secondary structure, which is nearly identical in many mitochondrial introns. The conserved secondary structure may be required for intron splicing. The second intron contains an open reading frame, continuous with the preceding exon, of approximately 290 codons. Two stretches of 10 amino acid residues, conserved in many introns, are present in the open reading frame.     

Comparative and functional anatomy of group II catalytic introns--a review

The 70 published sequences of group II introns from fungal and plant mitochondria and plant chloroplasts are analyzed for conservation of primary sequence, secondary structure and three-dimensional base pairings. Emphasis is put on structural elements with known or suspected functional significance with respect to self-splicing: the exon-binding and intron-binding sites, the bulging A residue involved in lariat formation, structural domain V and two isolated base pairs, one of them involving the last intron nucleotide and the other one, the first nt of the 3' exon. Separate sections are devoted to the 29 group II-like introns from Euglena chloroplasts and to the possible relationship of catalytic group II introns to nuclear premessenger introns. Alignments of all available sequences of group II introns are provided in the APPENDIX.