The recent origins of spliceosomal introns revisited

Does the intron/exon structure of eukaryotic genes belie their ancient assembly by exon-shuffling or have introns been inserted into preformed genes during eukaryotic evolution? These are the central questions in the ongoing ‘introns-early’ versus ‘introns-late’ controversy. The phylogenetic distribution of spliceosomal introns continues to strongly favor the intronslate theory. The introns-early theory, however, has claimed support from intron phase and protein structure correlations.     

Conserved intron positions in ancient protein modules

Background  

The timing of the origin of introns is of crucial importance for an understanding of early genome architecture. The Exon theory of genes proposed a role for introns in the formation of multi-exon proteins by exon shuffling and predicts the presence of conserved splice sites in ancient genes. In this study, large-scale analysis of potential conserved splice sites was performed using an intron-exon database (ExInt) derived from GenBank.     

Origin of new genes: evidence from experimental and computational analyses

Exon shuffling is an essential molecular mechanism for the formation of new genes. Many cases of exon shuffling have been reported in vertebrate genes. These discoveries revealed the importance of exon shuffling in the origin of new genes. However, only a few cases of exon shuffling were reported from plants and invertebrates, which gave rise to the assertion that the intron-mediated recombination mechanism originated very recently. We focused on the origin of new genes by exon shuffling and retroposition. We will first summarize our experimental work, which revealed four new genes in Drosophila, plants, and humans. These genes are 10⁶ to 10⁸ million years old. The recency of these genes allows us to directly examine the origin and evolution of genes in detail. These observations show firstly the importance of exon shuffling and retroposition in the rapid creation of new gene structures. They also show that the resultant chimerical structures appearing as mosaic proteins or as retroposed coding structures with novel regulatory systems, often confer novel functions. Furthermore, these newly created genes appear to have been governed by positive Darwinian selection throughout their history, with rapid changes of amino acid sequence and gene structure in very short periods of evolution. We further analyzed the distribution of intron phases in three non-vertebrate species, Drosophila melanogaster, Caenorhabditis elegans, and Arabidosis thaliana, as inferred from their genome sequences. As in the case of vertebrate genes, we found that intron phases in these species are unevenly distributed with an excess of phase zero introns and a significant excess of symmetric exons. Both findings are consistent with the requirements for the molecular process of exon shuffling. Thus, these non-vertebrate genomes may have also been strongly impacted by exon shuffling in general.     

Association of intron phases with conservation at splice site sequences and evolution of spliceosomal introns.

How exon-intron structures of eukaryotic genes evolved under various evolutionary forces remains unknown. The phases of spliceosomal introns (the placement of introns with respect to reading frame) provide an opportunity to approach this question. When a large number of nuclear introns in protein-coding genes were analyzed, it was found that most introns were of phase 0, which keeps codons intact. We found that the phase distribution of spliceosomal introns is strongly correlated with the sequence conservation of splice signals in exons; the relatively underrepresented phase 2 introns are associated with the lowest conservation, the relatively overrepresented phase 0 introns display the highest conservation, and phase 1 introns are intermediate. Given the detrimental effect of mutations in exon sequences near splice sites as found in molecular experiments, the underrepresentation of phase 2 introns may be the result of deleterious-mutation-driven intron loss, suggesting a possible genetic mechanism for the evolution of intron-exon structures.     

Centripetal modules and ancient introns

We have created an algorithm which instantiates the centripetal definition of modules, compact regions of protein structure, as introduced by Go and Nosaka (M. Go and M. Nosaka, 1987. Protein architecture and the origin of introns. Cold Spring Harbor Symp. Quant. Bio. 52, 915-924). That definition seeks the minima of a function that sums the squares of C-alpha carbon distances over a window around each amino acid residue in a three-dimensional protein structure and identifies such minima with module boundaries. We analyze a set of 44 ancient conserved proteins, with known three-dimensional structures, which have intronless homologues in bacteria and intron-containing homologues in the eukaryotes, with a corresponding set of 988 intron positions. We show that the phase zero intron positions are significantly correlated with the module boundaries (p = 0.0002), while the intron positions that lie within codons, in phase one and phase two, are not correlated with these 'centripetal' module boundaries. Furthermore, we analyze the phylogenetic distribution of intron positions and identify a subset of putatively 'ancient' intron positions: phase zero positions in one phylogenetic kingdom which have an associated intron either in an identical position or within three codons in another phylogenetic kingdom (a notion of intron sliding). This subset of 120 'ancient' introns lies closer to the module boundaries than does the full set of phase zero introns with high significance, a p-value of 0.008. We conclude that the behavior of this set of introns supports the prediction of a mixed theory: that some introns are very old and were used for exon shuffling in the progenote, while many introns have been lost and added since.     

Evolutionary history of exon shuffling

Exon shuffling has been characterized as one of the major evolutionary forces shaping both the genome and the proteome of eukaryotes. This mechanism was particularly important in the creation of multidomain proteins during animal evolution, bringing a number of functional genetic novelties. Here, genome information from a variety of eukaryotic species was used to address several issues related to the evolutionary history of exon shuffling. By comparing all protein sequences within each species, we were able to characterize exon shuffling signatures throughout metazoans. Intron phase (the position of the intron regarding the codon) and exon symmetry (the pattern of flanking introns for a given exon or block of adjacent exons) were features used to evaluate exon shuffling. We confirmed previous observations that exon shuffling mediated by phase 1 introns (1-1 exon shuffling) is the predominant kind in multicellular animals. Evidence is provided that such pattern was achieved since the early steps of animal evolution, supported by a detectable presence of 1-1 shuffling units in Trichoplax adhaerens and a considerable prevalence of them in Nematostella vectensis. In contrast, Monosiga brevicollis, one of the closest relatives of metazoans, and Arabidopsis thaliana, showed no evidence of 1-1 exon or domain shuffling above what it would be expected by chance. Instead, exon shuffling events are less abundant and predominantly mediated by phase 0 introns (0-0 exon shuffling) in those non-metazoan species. Moreover, an intermediate pattern of 1-1 and 0-0 exon shuffling was observed for the placozoan T. adhaerens, a primitive animal. Finally, characterization of flanking intron phases around domain borders allowed us to identify a common set of symmetric 1-1 domains that have been shuffled throughout the metazoan lineage.     

Recent evidence for the exon theory of genes

I provide a history of the Exon Theory of Genes, a theory that holds that introns are extremely ancient characteristics of genes and that early genes were created through the intron-mediated shuffling of exons. The theory has existed since the late seventies. An uncompromising version of the theory, in which all introns were considered to be ancient, dominated the early work. However, in the late eighties and early nineties evidence arose that at least some introns are more recently acquired. Due to the previously overly polarized nature of the debate, many results that demonstrated the existence of recent introns have been interpreted by some as evidence that all introns are late, leading some to believe that the theory is no longer a viable theory. However, recent work emphasizes a mixed model, in which some introns are ancient and some new. This model has proven to provide impressive explanatory power for a broad array of observations. Thus, this moderated form of the Exon Theory of Genes still offers a coherent picture of the origin and evolutionary history of the intron–exon structure of eukaryotic genes.     

Analysis of nonuniformity in intron phase distribution.

The distribution of different intron groups with respect to phases has been analyzed. It has been established that group II introns and nuclear introns have a minimum frequency of phase 2 introns. Since the phase of introns is an extremely conservative measure the observed minimum reflects evolutionary processes. A sample of all known, group I introns was too small to provide a valid characteristic of their phase distribution. The findings observed for the unequal distribution of phases cannot be explained solely on the basis of the mobile properties of introns. One of the most likely explanations for this nonuniformity in the intron phase distribution is the process of exon shuffling. It is proposed that group II introns originated at the early stages of evolution and were involved in the process of exon shuffling.     

Intron-exon structures of eukaryotic model organisms.

To investigate the distribution of intron-exon structures of eukaryotic genes, we have constructed a general exon database comprising all available intron-containing genes and exon databases from 10 eukaryotic model organisms: Homo sapiens, Mus musculus, Gallus gallus, Rattus norvegicus, Arabidopsis thaliana, Zea mays, Schizosaccharomyces pombe, Aspergillus, Caenorhabditis elegans and Drosophila. We purged redundant genes to avoid the possible bias brought about by redundancy in the databases. After discarding those questionable introns that do not contain correct splice sites, the final database contained 17 102 introns, 21 019 exons and 2903 independent or quasi-independent genes. On average, a eukaryotic gene contains 3.7 introns per kb protein coding region. The exon distribution peaks around 30-40 residues and most introns are 40-125 nt long. The variable intron-exon structures of the 10 model organisms reveal two interesting statistical phenomena, which cast light on some previous speculations. (i) Genome size seems to be correlated with total intron length per gene. For example, invertebrate introns are smaller than those of human genes, while yeast introns are shorter than invertebrate introns. However, this correlation is weak, suggesting that other factors besides genome size may also affect intron size. (ii) Introns smaller than 50 nt are significantly less frequent than longer introns, possibly resulting from a minimum intron size requirement for intron splicing.     

Molecular evolution of calmodulin-like domain protein kinases (CDPKs) in plants and protists

Many genes for calmodulin-like domain protein kinases (CDPKs) have been identified in plants and Alveolate protists. To study the molecular evolution of the CDPK gene family, we performed a phylogenetic analysis of CDPK genomic sequences. Analysis of introns supports the phylogenetic analysis; CDPK genes with similar intron/exon structure are grouped together on the phylogenetic tree. Conserved introns support a monophyletic origin for plant CDPKs, CDPK-related kinases, and phosphoenolpyruvate carboxylase kinases. Plant CDPKs divide into two major branches. Plant CDPK genes on one branch share common intron positions with protist CDPK genes. The introns shared between protist and plant CDPKs presumably originated before the divergence of plants from Alveolates. Additionally, the calmodulin-like domains of protist CDPKs have intron positions in common with animal and fungal calmodulin genes. These results, together with the presence of a highly conserved phase zero intron located precisely at the beginning of the calmodulin-like domain, suggest that the ancestral CDPK gene could have originated from the fusion of protein kinase and calmodulin genes facilitated by recombination of ancient introns. Received: 11 July 2000 / Accepted: 18 April 2001     

Evidence for the late origin of introns in chloroplast genes from an evolutionary analysis of the genus Euglena.

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.     

Transposition and exon shuffling by group II intron RNA molecules in pieces

In the realms of RNA, transposable elements created by self-inserting introns recombine novel combinations of exon sequences in the background of replicating molecules. Although intermolecular RNA recombination is a wide-spread phenomenon reported for a variety of RNA-containing viruses, direct evidence to support the theory that modern splicing systems, together with the exon-intron structure, have evolved from the ability of RNA to recombine, is lacking. Here, we used an in vitro deletion-complementation assay to demonstrate trans-activation of forward and reverse self-splicing of a fragmented derivative of the group II intron bI1 from yeast mitochondria. We provide direct evidence for the functional interchangeability of analogous but non-identical domain 1 RNA molecules of group II introns that result in trans-activation of intron transposition and RNA-based exon shuffling. The data extend theories on intron evolution and raise the intriguing possibility that naturally fragmented group III and spliceosomal introns themselves can create transposons, permitting rapid evolution of protein-coding sequences by splicing reactions.     

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.     

Analysis of ribosomal protein gene structures: implications for intron evolution

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

Protein domains correlate strongly with exons in multiple eukaryotic genomes--evidence of exon shuffling?

We conducted a multi-genome analysis correlating protein domain organization with the exon-intron structure of genes in nine eukaryotic genomes. We observed a significant correlation between the borders of exons and domains on a genomic scale for both invertebrates and vertebrates. In addition, we found that the more complex organisms displayed consistently stronger exon-domain correlation, with substantially more significant correlations detected in vertebrates compared with invertebrates. Our observations concur with the principles of exon shuffling theory, including the prediction of predominantly symmetric phase of introns flanking the borders of correlating exons. These results suggest that extensive exon shuffling events during evolution significantly contributed to the shaping of eukaryotic proteomes.     

Signs of Ancient and Modern Exon-Shuffling Are Correlated to the Distribution of Ancient and Modern Domains Along Proteins

Exon-shuffling is an important mechanism accounting for the origin of many new proteins in eukaryotes. However, its role in the creation of proteins in the ancestor of prokaryotes and eukaryotes is still debatable. Excess of symmetric exons is thought to represent evidence for exon-shuffling since the exchange of exons flanked by introns of the same phase does not disrupt the reading frame of the host gene. In this report, we found that there is a significant correlation between symmetric units of shuffling and the age of protein domains. Ancient domains, present in both prokaryotes and eukaryotes, are more frequently bounded by phase 0 introns and their distribution is biased towards the central part of proteins. Modern domains are more frequently bounded by phase 1 introns and are present predominantly at the ends of proteins. We propose a model in which shuffling of ancient domains mainly flanked by phase 0 introns was important in the ancestor of eukaryotes and prokaryotes, during the creation of the central part of proteins. Shuffling of modern domains, predominantly flanked by phase 1 introns, accounted for the origin of the extremities of proteins during eukaryotic evolution.     

A glimpse of a putative pre-intron phase of eukaryotic evolution

Comparison of the exon-intron structures of ancient eukaryotic paralogs reveals the absence of conserved intron positions in these genes. This is in contrast to the conservation of intron positions in orthologous genes from even the most evolutionarily distant eukaryotes and in more recent paralogs. The lack of conserved intron positions in ancient paralogs probably reflects the origination of these genes during the earliest phase of eukaryotic evolution, which was characterized by concomitant invasion of genes by group II self-splicing elements (which were to become introns in the future) and extensive duplication of genes.     

Module-intron correlation and intron sliding in family F/10 xylanase genes

Xylanases are classified into two families, numbered F/10 and G/11 according to the similarity of amino acid sequences of their catalytic domain (Henrissat, B., Bairoch, A., 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293, 781-788). Three-dimensional structure of the catalytic domain of the family F/10 xylanase was reported (White, A., Withers, S.G., Gilkes, N.R., Rose, D.R., 1994. Crystal structure of the catalytic domain of the beta-1,4-glycanase Cex from Cellulomonas fimi. Biochemistry 33, 12546-12552). The domain was decomposed into 22 modules by centripetal profiles (Go, M., Nosaka, M., 1987. Protein architecture and the origin of introns. Cold Spring Harbor Symp. Quant. Biol. 52, 915-924; Noguti, T., Sakakibara, H., Go, M., 1993. Localization of hydrogen-bonds within modules in barnase. Proteins 16, 357-363). A module is a contiguous polypeptide segment of amino acid residues having a compact conformation within a globular domain. Collected 31 intron sites of the family F/10 xylanase genes from fungus were found to be correlated to module boundaries with considerable statistical force (p values <0.001). The relationship between the intron locations and protein structures provides supporting evidence for the ancient origin of introns, because such a relationship cannot be expected by random insertion of introns into eukaryotic genes, but it rather suggests pre-existence of introns in the ancestral genes of prokaryotes and eukaryotes. A phylogenetic tree of the fungal and bacterial xylanase sequences made two clusters; one includes both the bacterial and fungal genes, but the other consists of only fungal genes. The mixed cluster of bacterial genes without introns and the fungal genes with introns further supports the ancient origin of introns. Comparison of the conserved base sequences of introns indicates that sliding of a splice site occurred in Aspergillus kawachii gene by one base from the ancestral position. Substrate-binding sites of xylanase are localized on eight modules, and introns are found at both termini of six out of these functional modules. This result suggests that introns might play a functional role in shuffling the exons encoding the substrate-binding modules.     

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