A possible role of exon-shuffling in the evolution of signal peptides of human proteins

It was recently shown that there is a predominance of phase 1 introns near the cleavage site of signal peptides encoded by human genes. It was suggested that this biased distribution was due to intron insertion at AGmid R:G proto-splice sites. However, we found that there is no disproportional excess of AGmid R:G that would support insertion at proto-splice sites. In fact, all nGmid R:G sites are enriched in the vicinity of the cleavage site. Additional analyses support an alternative scenario in which exon-shuffling is largely responsible for such excess of phase 1 introns.     

Evidence of splice signal migration from exon to intron during intron evolution

A comparison of the nucleotide sequences around the splice junctions that flank old (shared by two or more major lineages of eukaryotes) and new (lineage-specific) introns in eukaryotic genes reveals substantial differences in the distribution of information between introns and exons. Old introns have a lower information content in the exon regions adjacent to the splice sites than new introns but have a corresponding higher information content in the intron itself. This suggests that introns insert into nonrandom (proto-splice) sites but, during the evolution of an intron after insertion, the splice signal shifts from the flanking exon regions to the ends of the intron itself. Accumulation of information inside the intron during evolution suggests that new introns largely emerge de novo rather than through propagation and migration of old introns.     

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.     

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.     

Spliceosomal intron insertions in genome compacted ray-finned fishes as evident from phylogeny of MC receptors, also supported by a few other GPCRs

Background

Insertions of spliceosomal introns are very rare events during evolution of vertebrates and the mechanisms governing creation of novel intron(s) remain obscure. Largely, gene structures of melanocortin (MC) receptors are characterized by intron-less architecture. However, recently a few exceptions have been reported in some fishes. This warrants a systematic survey of MC receptors for understanding intron insertion events during vertebrate evolution.

Methodology/Principal Findings

We have compiled an extended list of MC receptors from different vertebrate genomes with variations in fishes. Notably, the closely linked MC2Rs and MC5Rs from a group of ray-finned fishes have three and one intron insertion(s), respectively, with conserved positions and intron phase. In both genes, one novel insertion was in the highly conserved DRY motif at the end of helix TM3. Further, the proto-splice site MAG↑R is maintained at intron insertion sites in these two genes. However, the orthologs of these receptors from zebrafish and tetrapods are intron-less, suggesting these introns are simultaneously created in selected fishes. Surprisingly, these novel introns are traceable only in four fish genomes. We found that these fish genomes are severely compacted after the separation from zebrafish. Furthermore, we also report novel intron insertions in P2Y receptors and in CHRM3. Finally, we report ultrasmall introns in MC2R genes from selected fishes.

Conclusions/Significance

The current repository of MC receptors illustrates that fishes have no MC3R ortholog. MC2R, MC5R, P2Y receptors and CHRM3 have novel intron insertions only in ray-finned fishes that underwent genome compaction. These receptors share one intron at an identical position suggestive of being inserted contemporaneously. In addition to repetitive elements, genome compaction is now believed to be a new hallmark that promotes intron insertions, as it requires rapid DNA breakage and subsequent repair processes to gain back normal functionality.     

Proto-splice site model of intron origin

It is proposed that nuclear pre-mRNA introns (classical introns) were first generated as by-products during the evolution of alternative splicing. They were formed whenever two splice sites within the coding sequence of ancestral genes were used at a frequency that removed the coding constraint from the intervening sequence. Once introns had evolved, it is suggested that they were spread by the splicing machinery which inserted them into proto or cryptic-splice sites of other genes by reverse splicing, so giving rise to genes that have introns yet are not alternatively spliced. It is argued that 5' and 3' splice sites evolved from common ancestral splice sites, referred to as proto-splice sites, that were bidirectional and had a core consensus sequence of C or A, A, G, R, which remains today as the immediate flanking sequence of most introns. The ancestral splicing machinery, although inefficient, would have been capable of generating vast mRNA diversity by splicing between proto-splice sites. Natural selection would be expected to have preserved mutations that increased the amounts of advantageously spliced mRNA. It is argued that this process drove the evolution of present 5' and 3' splice sites from a subset of proto-splice sites and also drove the evolution of a more efficient splicing machinery. The positions of most introns that evolved directly from the coding sequence would be expected to correlate with protein structure.     

Insertion of spliceosomal introns in proto-splice sites: the case of secretory signal peptides

Analysis of the exon-intron structures of 2208 human genes has revealed that there is a statistically highly significant excess of phase 1 introns in the vicinity of the signal peptide cleavage sites. It is suggested that amino acid sequences surrounding signal peptide cleavage sites are significantly enriched in phase 1 proto-splice sites and this has favored insertion of spliceosomal introns in these sites.     

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.     

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.     

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.     

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.     

Does Drive Toward Canonic Exonic Splicing Sites Exist in Mammals?

About 2/3 of introns are inserted between G and G/A, which has previously been explained by codon usage frequencies existing during the period of intron insertions. However, less is known about the evolution of exonic splicing sites. Exonic nucleotides that frame introns are involved in both protein coding and splicing. While a compromise between protein coding and splicing constraints is achieved differently in each intron phase, AG|G is the most common site in all phases comprising about one quarter of all such sites. There is also a great variety of other splicing sites. Here we examine evolutionary changes in exonic nucleotides located at positions −2 −1|+1 which occurred after the beginning of eutherian radiation using comparisons of orthologous splicing sites from five mammalian species. AG|G accumulated fewer substitutions and was more conservative than less frequent exonic splicing sites. Such trend could potentially increase frequencies of AG|G during mammalian evolution and cause a decline of less common sites which had higher substitution rates. However, there is a limit to this process determined by the dynamic equilibrium of substitution rates and the frequencies of different splicing sites. It seems that this equilibrium was already achieved at the time of eutherian radiation and a moderate increase in AG|G frequency was observed only in the human genome.     

Can Codon Usage Bias Explain Intron Phase Distributions and Exon Symmetry?

More introns exist between codons (phase 0) than between the first and the second bases (phase 1) or between the second and the third base (phase 2) within the codon. Many explanations have been suggested for this excess of phase 0. It has, for example, been argued to reflect an ancient utility for introns in separating exons that code for separate protein modules. There may, however, be a simple, alternative explanation. Introns typically require, for correct splicing, particular nucleotides immediately 5 in exons (typically a G) and immediately 3 in the following exon (also often a G). Introns therefore tend to be found between particular nucleotide pairs (e.g., G|G pairs) in the coding sequence. If, owing to bias in usage of different codons, these pairs are especially common at phase 0, then intron phase biases may have a trivial explanation. Here we take codon usage frequencies for a variety of eukaryotes and use these to generate random sequences. We then ask about the phase of putative intron insertion sites. Importantly, in all simulated data sets intron phase distribution is biased in favor of phase 0. In many cases the bias is of the magnitude observed in real data and can be attributed to codon usage bias. It is also known that exons may carry either the same phase (symmetric) or different phases (asymmetric) at the opposite ends. We simulated a distribution of different types of exons using frequencies of introns observed in real genes assuming random combination of intron phases at the opposite sides of exons. Surprisingly the simulated pattern was quite similar to that observed. In the simulants we typically observe a prevalence of symmetric exons carrying phase 0 at both ends, which is common for eukaryotic genes. However, at least in some species, the extent of the bias in favor of symmetric (0,0) exons is not as great in simulants as in real genes. These results emphasize the need to construct a biologically relevant null model of successful intron insertion.Reviewing Editor: Dr. Manyuan Long     

Selective constraints on intron evolution in Drosophila

Intron sizes show an asymmetrical distribution in a number of organisms, with a large number of "short" introns clustered around a minimal intron length and a much broader distribution of longer introns. In Drosophila melanogaster, the short intron class is centered around 61 bp. The narrow length distribution suggests that natural selection may play a role in maintaining intron size. A comparison of 15 orthologous introns among species of the D. melanogaster subgroup indicates that, in general, short introns are not under greater DNA sequence or length constraints than long introns. There is a bias toward deletions in all introns (deletion/insertion ratio is 1.66), and the vast majority of indels are of short length (<10 bp). Indels occurring on the internal branches of the phylogenetic tree are significantly longer than those occurring on the terminal branches. These results are consistent with a compensatory model of intron length evolution in which slightly deleterious short deletions are frequently fixed within species by genetic drift, and relatively rare larger insertions that restore intron length are fixed by positive selection. A comparison of paralogous introns shared among duplicated genes suggests that length constraints differ between introns within the same gene. The janusA, janusB, and ocnus genes share two short introns derived from a common ancestor. The first of these introns shows significantly fewer indels than the second intron, although the two introns show a comparable number of substitutions. This indicates that intron-specific selective constraints have been maintained following gene duplication, which preceded the divergence of the D. melanogaster species subgroup.     

A very high fraction of unique intron positions in the intron-rich diatom Thalassiosira pseudonana indicates widespread intron gain

Although spliceosomal introns are present in all characterized eukaryotes, intron numbers vary dramatically, from only a handful in the entire genomes of some species to nearly 10 introns per gene on average in vertebrates. For all previously studied intron-rich species, significant fractions of intron positions are shared with other widely diverged eukaryotes, indicating that 1) large numbers of the introns date to much earlier stages of eukaryotic evolution and 2) these lineages have not passed through a very intron-poor stage since early eukaryotic evolution. By the same token, among species that have lost nearly all of their ancestral introns, no species is known to harbor large numbers of more recently gained introns. These observations are consistent with the notion that intron-dense genomes have arisen only once over the course of eukaryotic evolution. Here, we report an exception to this pattern, in the intron-rich diatom Thalassiosira pseudonana. Only 8.1% of studied T. pseudonana intron positions are conserved with any of a variety of divergent eukaryotic species. This implies that T. pseudonana has both 1) lost nearly all of the numerous introns present in the diatom-apicomplexan ancestor and 2) gained a large number of new introns since that time. In addition, that so few apparently inserted T. pseudonana introns match the positions of introns in other species implies that insertion of multiple introns into homologous genic sites in eukaryotic evolution is less common than previously estimated. These results suggest the possibility that intron-rich genomes may have arisen multiple times in evolution. These results also provide evidence that multiple intron insertion into the same site is rare, further supporting the notion that early eukaryotic ancestors were very intron rich.     

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.     

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.     

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.