The evolution of spliceosomal introns

Although the widespread proliferation of introns in eukaryotic protein-coding genes remains one of the most poorly understood aspects of genomic architecture, major advances have emerged recently from large-scale genome sequencing projects and functional analyses of mRNA-processing events. Evidence supports the idea that spliceosomal introns were not only present in the stem eukaryote but diverged into at least two distinct classes very early in eukaryotic evolution. Some rough estimates of intron turnover rates are provided, and a testable hypothesis for the origin of new introns is proposed. In light of recent findings on the molecular natural history of splicing, various aspects of the phylogenetic and physical distributions of introns can now be interpreted in a theoretical framework that jointly considers the population-genetic roles of mutation, random genetic drift, and natural selection.     

Phase distribution of spliceosomal introns: implications for intron origin

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.     

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.     

The evolution of spliceosomal introns in alveolates

Many issues concerning the evolution of spliceosomal introns remain poorly understood. In this respect, the reconstruction of the evolution of introns in deep branching species such as alveolates is of special significance. In this study, we inferred the intron evolution in alveolates using 3,368 intron positions in 162 orthologs from 10 species (9 alveolates and 1 outgroup, Homo sapiens). We found that although very few intron gains and losses have occurred in Theileria and Plasmodium recently, many intron gains and losses have occurred in the evolution of alveolates. Thus, the rates of intron gain and loss in alveolates have varied greatly across time and lineage. Our results seem to support the notion that massive intron gains and losses have occurred during short episodes, perhaps coinciding with major evolutionary events.     

Origin and evolution of spliceosomal introns

ABSTRACT: Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded 'introns first' held 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. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns. Thus, the introns-first scenario is not supported by any evidence but exon-intron structure of protein-coding genes appears to have evolved concomitantly with the eukaryotic cell, and introns were a major factor of evolution throughout the history of eukaryotes. This article was reviewed by I. King Jordan, Manuel Irimia (nominated by Anthony Poole), Tobias Mourier (nominated by Anthony Poole), and Fyodor Kondrashov. For the complete reports, see the Reviewers' Reports section.     

U12-type spliceosomal introns of Insecta

Most of eukaryotic genes are interrupted by introns that need to be removed from pre-mRNAs before they can perform their function. This is done by complex machinery called spliceosome. Many eukaryotes possess two separate spliceosomal systems that process separate sets of introns. The major (U2) spliceosome removes majority of introns, while minute fraction of intron repertoire is processed by the minor (U12) spliceosome. These two populations of introns are called U2-type and U12-type, respectively. The latter fall into two subtypes based on the terminal dinucleotides. The minor spliceosomal system has been lost independently in some lineages, while in some others few U12-type introns persist. We investigated twenty insect genomes in order to better understand the evolutionary dynamics of U12-type introns. Our work confirms dramatic drop of U12-type introns in Diptera, leaving these genomes just with a handful cases. This is mostly the result of intron deletion, but in a number of dipteral cases, minor type introns were switched to a major type, as well. Insect genes that harbor U12-type introns belong to several functional categories among which proteins binding ions and nucleic acids are enriched and these few categories are also overrepresented among these genes that preserved minor type introns in Diptera.     

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.     

The evolution of spliceosomal introns: patterns, puzzles and progress

The origins and importance of spliceosomal introns comprise one of the longest-abiding mysteries of molecular evolution. Considerable debate remains over several aspects of the evolution of spliceosomal introns, including the timing of intron origin and proliferation, the mechanisms by which introns are lost and gained, and the forces that have shaped intron evolution. Recent important progress has been made in each of these areas. Patterns of intron-position correspondence between widely diverged eukaryotic species have provided insights into the origins of the vast differences in intron number between eukaryotic species, and studies of specific cases of intron loss and gain have led to progress in understanding the underlying molecular mechanisms and the forces that control intron evolution.     

Evolutionary dynamics of U12-type spliceosomal introns

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.     

Evolution of spliceosomal introns following endosymbiotic gene transfer

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.     

Genome-wide analysis of recombination machinery for spliceosomal introns gain

What caused spliceosomal introns gain remains an unsolved problem. To this, defining what spliceosomal introns arise from is critical. Here, the introns density of the genomes is calculated for four species, indicating:(1) sex chromosomes in mammals have lower intron densities, (2) despite that, the proportion of UTRs (untranslated regions) with introns in sex chromosomes is higher than other ones, and (3) AT content of introns is more similar to that of intergenic regions when these regions comprise the majority of a chromosome, and more similar to that of exons, when exons are the majority of the chromosome. On the other hand, introns have been clearly demonstrated to invade genetic sequences in recent times while sex chromosomes evolved from a pair of autosomes within the last 300 millions years. One main difference between sex chromosomes and autosomes in mammalian is that sex chromosomes recombination stopped. Thus, recombination might be the main determinant for eukaryotes gaining spliceosomal introns. To further prove that and avoid giving weak signal, the whole genomes from eight eukaryotic species are analyzed and present strong signal for above the trend (3) in three species (t-test, P = 0.55 for C. elegans, P = 0.72 for D. melanogaster and P = 0.83 for A. thaliana). These results suggest that the genome-wide coincidence as above (3) can only be caused by the large-scale random unequal crossover in eukaryote meiosis, which might have fueled spliceosomal introns but hardly occurred in prokaryotes.     

A high density of ancient spliceosomal introns in oxymonad excavates

Background  

Certain eukaryotic genomes, such as those of the amitochondriate parasites Giardia and Trichomonas, have very low intron densities, so low that canonical spliceosomal introns have only recently been discovered through genome sequencing. These organisms were formerly thought to be ancient eukaryotes that diverged before introns originated, or at least became common. Now however, they are thought to be members of a supergroup known as excavates, whose members generally appear to have low densities of canonical introns. Here we have used environmental expressed sequence tag (EST) sequencing to identify 17 genes from the uncultivable oxymonad Streblomastix strix, to survey intron densities in this most poorly studied excavate group.     

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.     

Widespread occurrence of spliceosomal introns in the rDNA genes of ascomycetes

Spliceosomal (pre-mRNA) introns have previously been found in eukaryotic protein-coding genes, in the small nuclear RNAs of some fungi, and in the small- and large-subunit ribosomal DNA genes of a limited number of ascomycetes. How the majority of these introns originate remains an open question because few proven cases of recent and pervasive intron origin have been documented. We report here the widespread occurrence of spliceosomal introns (69 introns at 27 different sites) in the small- and large-subunit nuclear-encoded rDNA of lichen-forming and free-living members of the Ascomycota. Our analyses suggest that these spliceosomal introns are of relatively recent origin, i.e., within the Euascomycetes, and have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into rRNAs. The spliceosome itself, and not an external agent (e.g., transposable elements, group II introns), may have given rise to these introns. A nonrandom sequence pattern was found at sites flanking the rRNA spliceosomal introns. This pattern (AG-intron-G) closely resembles the proto-splice site (MAG-intron-R) postulated for intron insertions in pre-mRNA genes. The clustered positions of spliceosomal introns on secondary structures suggest that particular rRNA regions are preferred sites for insertion through reverse-splicing.     

Birth of new spliceosomal introns in fungi by multiplication of introner-like elements

Spliceosomal introns are noncoding sequences that separate exons in eukaryotic genes and are removed from pre-messenger RNAs by the splicing machinery. Their origin has remained a mystery in biology since their discovery because intron gains seem to be infrequent in many eukaryotic lineages. Although a few recent intron gains have been reported, none of the proposed gain mechanisms can convincingly explain the high number of introns in present-day eukaryotic genomes. Here we report on particular spliceosomal introns that share high sequence similarity and are reminiscent of introner elements. These elements multiplied in unrelated genes of six fungal genomes and account for the vast majority of intron gains in these fungal species. Such introner-like elements (ILEs) contain all typical characteristics of regular spliceosomal introns (RSIs) but are longer and predicted to harbor more stable secondary structures. However, dating of multiplication events showed that they degenerate in sequence and length within 100,000 years to eventually become indistinguishable from RSIs. We suggest that ILEs not only account for intron gains in six fungi but also in ancestral eukaryotes to give rise to most RSIs by a yet unknown multiplication mechanism.     

The abundance of the spliceosomal snRNPs is not limiting the splicing of U12-type introns

The rate of excision of U12-type introns has been reported to be slower than that of U2-type introns, suggesting a rate-limiting bottleneck that could down-regulate genes containing U12-type introns. The mechanistic reasons for this slower rate of intron excision are not known, but lower abundance of the U12-type snRNPs and slower rate of assembly or catalytic activity have been suggested. To investigate snRNP abundance we concentrated on the U4atac snRNA, which is the least abundant of the U12-type snRNAs and is limiting the formation of U4atac/U6atac complex. We identified mouse NIH-3T3 cell line isolates in which the level of both U4atac snRNA and U4atac/U6atac complexes is reduced to 10%-20% of the normal level. We used these cell lines to investigate splicing efficiency by transient transfection of a reporter gene containing a U12-type intron and by quantitative PCR analysis of endogenous genes. The splicing of the reporter U12-type intron was very inefficient, but the activity could be restored by overexpression of U4atac snRNA. Using these U4atac-deficient NIH-3T3 cells, we confirmed the results of previous studies showing that U12-type introns of endogenous genes are, indeed, excised more slowly than U2-type introns, but we found that the rate did not differ from that measured in cells displaying normal levels of U4atac snRNA. Thus our results suggest that the cellular abundance of the snRNPs does not limit U12-type intron splicing under normal conditions.     

Retention of spliceosomal components along ligated exons ensures efficient removal of multiple introns

The majority of mammalian pre-mRNAs contains multiple introns that are excised prior to export and translation. After intron excision, ligated exon intermediates participate in subsequent intron excisions. However, exon ligation generates an exon of increased size, a feature of pre-mRNA splicing that can interfere with downstream splicing events. These considerations raise the question of whether unique mechanisms exist that permit efficient removal of introns neighboring ligated exons. Kinetic analyses of multiple intron-containing pre-mRNAs revealed that splicing is more efficient following an initial intron removal event, suggesting that either the recruitment of the exon junction complex (EJC) to ligated exons increases the efficiency of multiple intron excisions or that the initial definition of splice sites is sufficient to permit efficient splicing of introns neighboring ligated exons. Knockdown experiments show that the deposition of the EJC does not affect subsequent splicing kinetics. Instead, spliceosomal components that are not involved in the initial splicing event remain associated with the pre-mRNA to ensure efficient removal of neighboring introns. Thus, ligated exons do not require redefinition, providing an additional kinetic advantage for exon defined splice sites.     

Multiple gains of spliceosomal introns in a superfamily of vertebrate protease inhibitor genes

Background  

Intron gains reportedly are very rare during evolution of vertebrates, and the mechanisms underlying their creation are largely unknown. Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily genes were subject to massive changes, in contrast to many other genes.     

Splicing of a divergent subclass of AT-AC introns requires the major spliceosomal snRNAs.

AT-AC introns constitute a minor class of eukaryotic pre-mRNA introns, characterized by 5''-AT and AC-3'' boundaries, in contrast to the 5''-GT and AG-3'' boundaries of the much more prevalent conventional introns. In addition to the AT-AC borders, most known AT-AC introns have highly conserved 5'' splice site and branch site sequence elements of 7-8 nt. Intron 6 of the nucleolar P120 gene and intron 2 of the SCN4A voltage-gated skeletal muscle sodium channel are AT-AC introns that have been shown recently to be processed via a unique splicing pathway involving several minor U snRNAs. Interestingly, intron 21 of the same SCN4A gene and the corresponding intron 25 of the SCN5A cardiac muscle sodium channel gene also have 5''-AT and AC-3'' boundaries, but they have divergent 5'' splice site and presumptive branch site sequences. Here, we report the accurate in vitro processing of these two divergent AT-AC introns and show that they belong to a functionally distinct subclass of AT-AC introns. Splicing of these introns does not require U12, U4atac, and U6atac snRNAs, but instead requires the major spliceosomal snRNAs U1, U2, U4, U5, and U6. Previous studies showed that G --> A mutation at the first position and G --> C mutation at the last position of a conventional yeast or mammalian GT-AG intron suppress each other in vivo, suggesting that the first and last bases participate in an essential non-Watson-Crick interaction. Our results show that such introns, hereafter termed AT-AC II introns, occur naturally and are spliced by a mechanism distinct from that responsible for processing of the apparently more common AT-AC I introns.     

Domestication of self-splicing introns during eukaryogenesis: the rise of the complex spliceosomal machinery

?

The spliceosome is a eukaryote-specific complex that is essential for the removal of introns from pre-mRNA. It consists of five small nuclear RNAs (snRNAs) and over a hundred proteins, making it one of the most complex molecular machineries. Most of this complexity has emerged during eukaryogenesis, a period that is characterised by a drastic increase in cellular and genomic complexity. Although not fully resolved, recent findings have started to shed some light on how and why the spliceosome originated.In this paper we review how the spliceosome has evolved and discuss its origin and subsequent evolution in light of different general hypotheses on the evolution of complexity. Comparative analyses have established that the catalytic core of this ribonucleoprotein (RNP) complex, as well as the spliceosomal introns, evolved from self-splicing group II introns. Most snRNAs evolved from intron fragments and the essential Prp8 protein originated from the protein that is encoded by group II introns. Proteins that functioned in other RNA processes were added to this core and extensive duplications of these proteins substantially increased the complexity of the spliceosome prior to the eukaryotic diversification. The splicing machinery became even more complex in animals and plants, yet was simplified in eukaryotes with streamlined genomes. Apparently, the spliceosome did not evolve its complexity gradually, but in rapid bursts, followed by stagnation or even simplification. We argue that although both adaptive and neutral evolution have been involved in the evolution of the spliceosome, especially the latter was responsible for the emergence of an enormously complex eukaryotic splicing machinery from simple self-splicing sequences.

Reviewers

This article was reviewed by W. Ford Doolittle, Eugene V. Koonin and Vivek Anantharaman.