Department of Genetics, University of Leipzig, Leipzig, Germany
Correspondence: E-mail address: krauss{at}rz.uni-leipzig.de.
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Abstract |
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Key Words: eIF2 intron evolution molecular phylogenetics intron clustering arthropod phylogeny intron sliding
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Introduction |
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Among these singular character states, intron position appears to be rather unreliable (Krzywinski and Besansky 2002; Wada et al. 2002). Likely cases of intron insertion and loss have been documented (Rzhetsky et al. 1997; Logsdon, Stoltzfus, and Doolittle 1998; Feiber, Rangarajan, and Vaughn 2002; Roy, Fedorov, and Gilbert 2003; Brady and Danforth 2004). Based on recent genome projects, large-scale comparisons of intron positions has been done (Fedorov, Merican, and Gilbert 2002; Rogozin et al. 2003). Their results suggested that intron positioning is more dynamic than previously assumed. Therefore, comprehensive analyses of novel marker genes, focusing on both intron position and sequence data, would be useful.
We sought to analyze the evolution of sequence and exon-intron structure of a strongly conserved single-copy gene in a representative sample of eukaryotic species. For this purpose, we have chosen the subunit of eukaryotic translation initiation factor 2 (eIF2). By delivering the initiator methionyl-tRNA to the small subunit of ribosomes, eIF2 ensures specifity of initiation codon selection (Kapp and Lorsch 2004, Roll-Mecak et al. 2004). eIF2
is the strongest conserved subunit of the heterotrimeric eIF2 and is found in Eukaryota and Archaea.
In a preliminary study (Krauss and Reuter 2000), we described eIF2 gene structures of six arthropod species and showed that the gene is fused with the functionally unrelated Su(var)3-9 histone methyltransferase gene in holometabolic insects. Here, we have sequenced eIF2
genes of 13 other arthropod species and collected database sequences from 40 additional, selected eukaryotic species for our analysis. Examining the cladistic distribution of 52 different intron positions in 55 distantly related eIF2
genes, we identified ancient and shared derived introns. Our analysis has shown that intron positioning in eIF2
is evolutionarily conserved. However, there were episodes of complete or partial intron losses followed by intron gains. Using a maximum-parsimony analysis based on an intron presence/absence matrix, we showed that introns are phylogenetically informative. We note that in phylogenetic mapping of intron positions, sampling of taxa has to be as complete as possible.
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Materials and Methods |
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Isolation of eIF2 Genes Using PCR
DNA was isolated by standard protocols. Trizol reagent (Invitrogen) was used to isolate total RNA. cDNA was synthesized using Hminus-M-MLV reverse transcriptase (Fermentas) and a polyT primer. Degenerate primers based on the amino acid sequences of already known eIF2 proteins were designed to partially amplify the eIF2
gene from genomic DNA and/or cDNA of arthropod species. Used degenerated oligonucleotide primers were Ef120 (5'-CARGCXATHAAYATHGGXACXATHGGXCAYGTXGCXCAYGG-3'), Ef440 (5'-CCRTTXARCATXGTXSYCATXARDATRTCRTGXCCXGGRCARTC-3'), Ef120c (5'-AATATAGGAACCATTGGTCATGTNGCNCAYGG-3'), Ef440c (5'-TCCATCACAGCTGCTCCGTTCAACATNGTNGCCAT-3'), Efdeg3 (5'-GARCAYTTRGCSGCYATHGARATHATG-3'), Efdeg4 (5'-GCKTCKRCTSAGDGCWATYTTYTCKCC-3'), Efdeg5 (5'-ATTCGATCGTTYGAYGTVAAYAARCCNGG-3') and Efdeg6 (5'-TTTGTACCAACACCDATHARDCCNCCNGG-3'). Primer positions within eIF2
are shown in figure 1. PCR amplifications were done in a Gradient Cycler (Eppendorf) at annealing temperatures between 45°C and 65°C. The initial PCR product (320 bp to 900 bp) was purified using Spin PCRapid Kit (Macherey & Nagel) and sequenced. Species-specific primers were designed based on the received sequence to obtain 5' ends and 3' ends of eIF2
transcripts by 5' RACE (Rapid amplification of cDNA ends) and 3' RACE, respectively (GeneRacerKit, Invitrogen). Alternatively, inverted PCR products from digested and ligated genomic DNA preparations were purified, cloned, and sequenced. The specific sequencing strategy used for each of the analyzed species is given in figure 1 of Supplementary Material online. Species-specific primer sequences are available upon request.
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Sequence Sampling and Annotation
eIF2-orthologous DNA sequences from genome sequencing projects were sampled from databases using Blast. In particular, we used TBlastN (Altschul et al. 1997) based on nine already known eIF2
sequences (Krauss and Reuter 2000) to retrieve eIF2
-like genomic sequences from finished and unfinished genome projects deposited at the NCBI database. Additionally, single-trace sequences were screened at the TraceSite of NCBI (http://www.ncbi.nih.gov/blast/tracemb.html) using discontiguous MEGABlast and were assembled manually. Independently, we screened for similar EST sequences utilizing TBlastN and assembled these sequences if possible. The orthology of these candidate sequences was verified by multiple alignment and phylogenetic analysis. We excluded all angiosperm sequences, with the exception of Arabidopsis and Oryza eIF2
genes, from the sequence set because usage of incomplete angiosperm EST and genome data would complicate both gene assembling and phylogenetic analysis by frequent gene duplications. We also excluded all vertebrate sequences, with the exceptions of Homo and Takifugu, because protein identity between these species is exceedingly high (>95%), and we could not find any gene structure differences between vertebrate eIF2
genes.
Alignment and Mapping of Introns
Amino acid sequences were aligned using MacVector 7.2 (Accelrys) and revised by eye. The divergent ends of eIF2 proteins were deleted from the final data set. Intron positions at the corresponding nucleotide sequences were deduced by co-occurrence of splice consensus sites and gaps in similarity. All identified exon boundaries are supported by typical splice-site sequences of U2-dependent spliceosomal introns. This exon-intron structure was confirmed by cDNA sequence if available. Introns localized upstream or downstream from the conserved eIF2
ORF were not considered. We evaluated the location of introns with respect to (1) Drosophila melanogaster eIF2
amino acid residue numbering and (2) phase in ORF, which results in bipartite naming of all identified intron positions. Intron phase was named 0 if the intron splits two consecutive codons; 1 if an intron locates between the first and the second nucleotide of the codon; and 2 if an intron locates between the second and the third nucleotide of the codon.
Phylogenetic Analysis
The programs MrBayes version 3.0 (Ronquist and Huelsenbeck 2003), Tree-Puzzle version 5.0 (Schmidt et al. 2001), PAUP* version 4.0b10 (Swofford 2002), and MacVector 7.2 (Accelrys) were used for phylogenetic analyses. Tree constructions were performed through the Bayesian inference (BI) method by MrBayes using the JTT substitution model, 500,000 replicates (every 100th was saved), and a burn-in of 2,000, resulting in 3,000 trees. The posterior probability tree from this analysis was computed using PAUP*. For a maximum-likelihood (ML) analysis, we used quartet-puzzling by Tree-Puzzle 5.0 with 25,000 puzzling steps and the WAG substitution model, and we assumed rate heterogeneity with eight gamma rate categories. A maximum-parsimony (MP) analysis was done by heuristic bootstrapping (1,000 steps) using PAUP* and the branch-swapping algorithm tree-bisection-reconnection (TBR). Finally, a neighbor-joining (NJ) analysis was calculated by MacVector using bootstrapping (1,000 steps) and a Poisson-corrected distance.
We have utilized all intron positions of 37 genes in an independent tree reconstruction. Intronless genes were excluded from this analysis because a total erasure of introns from the eIF2 gene has taken place several times in parallel during evolution (see below). We built an input matrix based on presence/absence of a given intron and implemented a branch-and-bound search (MP) using PAUP* 4.0b10 (Swofford 2002). Characters were considered as unordered.
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Results and Discussion |
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eIF2 genes contain up to 11 introns in the conserved region of the ORF (Supplementary Material table 1), which ranges from amino acid alignment position 14 to position 477 in figure 1. Intronless as well as intron-rich eIF2
genes exist in species of each of the following taxa: protists, fungi, and deuterostomates (Supplementary Material table 1). It indicates that erasure of all introns from the gene structure (most probably by retrotransposition) has occurred several times independently during evolution. Nevertheless, eIF2
introns mapped onto multiple protein alignment show a remarkable conservation of intron locations.
An important initial assumption of our analysis is the homology of each specific intron position. We assume that introns might have only very rarely been gained at homologous sites in different evolutionary lineages, as implicated by the protosplice-site theory (Dibb and Newman 1989; Sadusky, Newman, and Dibb 2004), as compared with intron insertion at different sites. The strong conservation of the eIF2 protein sequence in the eukaryotic species (fig. 1) excludes alignment ambiguities resulting in wrong homologization or distinction of intron positions found in different species. Therefore, we considered only those intron positions as homologous that were identical in both location and phase.
Altogether, we found 52 different intron positions in eIF2 genes, and 22 of these introns were identified in only one of the analyzed species. The other 30 introns are present in two or more of those species and are very likely evolutionarily conserved. According to the intron-early theory, these introns predated the origin of eukaryotes and had an important role by assembling a functional protein from short-coding DNA sequences (Gilbert 1987). Thus, we mapped the location of exon boundaries in the tertiary structure described for two archaeal orthologs (fig. 1) (Schmitt, Blanquet, and Mechulam 2002; Roll-Mecak et al. 2004). eIF2
consists of three domains: G domain, domain II, and domain III. There is no local correlation of conserved intron positions with domain borders (fig. 1). We could not find any specific location of introns with respect to secondary structure elements. Furthermore, we noticed that the bacterial and mitochondrial protein EF-Tu, an elongation factor of translation, shows significant homology to all three domains of eIF2
in sequence and structure (Schmitt, Blanquet, and Mechulam 2002). Thus, all analyzed eIF2
genes likely evolved from an intronless ortholog. Accordingly, intron positioning in eIF2
has occurred independently from an eventual exon shuffling during early evolution. Hence, it follows that the pattern of eIF2
intron-exon boundaries should reveal suitable markers of eukaryotic phylogeny.
Distribution of Intron Positions and Exon Lengths
Next, we examined the exon length distribution in eIF2 genes (fig. 2A), which shows a maximum between 150 and 180 nt. This differs from an estimation of 90 to 120 nt based on a database of gene structures sampled from several model organisms (Deutsch and Long 1999). In agreement with this study, exons smaller than 60 nt are rare. A 16-nt exon between intron positions 39-0 and 44-1 was found in the related fungi Coprinus and Phanerochaete and a 23-nt exon between intron positions 133-1 and 141-0 was identified in the fungus Rhizopus. The rarity of small exons probably have some functional reasons. It was shown that exons shorter than 50 nt are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splice enhancers (Hwang and Cohen 1997; Carlo, Sierra, and Berget 2000). Thus, small exons should have evolved more seldom than the larger ones. In addition, such exons may occur more frequently in fungi than in other eukaryotes, which is consistent with the data of Deutsch and Long (1999).
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We examined the distribution of introns between the clusters. If different intron positions inside one cluster are homologous to each other, the abundance of introns found in each cluster should be independent from the number of different intron positions that belong to a cluster. However, we would expect a linear correlation of intron abundance to the number of intron positions in each cluster if each intron position were evolved independently. We found such a correlation (fig. 2C). An additional argument for evolutionary independence of each intron position is the rarity of intron sliding; that is, the movement of an existing intron to a nearby position (Stoltzfus et al. 1997; Rogozin, Lyons-Weiler, and Koonin 2000). Therefore, we suggest that apparent clusters of intron positions are mainly the result of intron erasure and subsequent insertion of novel introns at positions compatible with preferred exon sizes. A corresponding, relatively uniform exon size distribution was suggested to be based on functional limitations of the nonsense-mediated decay (NMD) pathway, which is involved in the cell's surveillance for transcripts harboring premature termination codons (Lynch and Richardson 2002; Lynch and Kewalramani 2003). Intron positions play a guiding role during the recognition of premature termination codons by NMD and, therefore, might have evolutionarily forced to a more uniform distribution than under a model of random insertion.
It appears that a clustered distribution of introns is not specific for the eIF2 gene. Wada et al. (2002) demonstrated at least one similar intron cluster (four different small shifts of their intron position 7) in deuterostome EF-1
genes. A very similar pattern of intron distribution was revealed in the insect chemoreceptor superfamily of Drosophila melanogaster (Robertson, Warr, and Carlson 2003).
Tree Analysis Based on eIF2 Gene Structure
We identified at least partial gene structures of 51 out of 55 analyzed eIF2 genes. Seven intronless genes and seven incompletely analyzed gene structures were excluded from the data set (table 1 in Supplementary Material online). An MP tree reconstruction (see Materials and Methods) was performed using the remaining 37 gene structures, represented by a presence/absence matrix including all intron positions (table 2 in Supplementary Material online). The resulting unrooted tree (fig. 3) shows a remarkable phylogenetic information content and supports, for example, the following monophyletic groups: Apicomplexa, Nematoda, Viridiplantae, Angiospermae, Deuterostomia, Homobasidiomycetes, and Pezizomycotina (Ascomycota sensu stricta). Other groupings are clearly spurious, such as the branching of the diatom Thalassiosira with nematodes or the branching of the flatworm Schistosoma with Coleomata. Interestingly, most representatives of Endopterygota (metamorphosing insects) did not group with the other arthropods. The common branching between those other arthropods (Daphnia, Apis, Aphis and Allacma), Schistosoma, and the represented deuterostomes can be explained by symplesiomorphic (shared ancient) intron positions (44-1, 127-2, 212-1, 394-0, and 451-2), probably acquired from the last common ancestor of all bilaterians, in their eIF2
genes. This finding may be related to results of recent studies (reviewed in Raible and Arendt [2004]) that revealed human and platworm genes seem to be closer to the bilaterian roots than are Drosophila and Caenorhabditis genes. This thesis is based on comparisons of gene content and similarity between orthologs. Our results point to a possibly slower evolution of gene structures in deuterostomes and some platworms as well. Additionally, arthropods appear to have evolved differentially fast in this respect.
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eIF2 Sequence Phylogeny
Phylogenetic analysis was carried out using BI, ML, MP, and NJ methods and an eIF2 amino acid sequence alignment (see Materials and Methods, and see figure 2 of Supplementary Material online). Preferably, a nucleotide sequence alignment was avoided because of high amounts of homoplasy, which would be expected from coding sequences separated by long divergence times. The aIF2
sequences of the archaeal species Pyrococcus abyssi and Methanococcus jannaschii were used as outgroups. The BI tree, which provides additional branching information from the other trees, is presented (fig. 4). Interesting results of these analyses are (1) the significant support of the Coelomata hypothesis in contrast to the Ecdysozoa hypothesis and (2) the sister-relationship between Daphnia and Allacma. The last result might support a novel phylogenetic hypothesis (Nardi et al. 2003). Accordingly, hexapods are not monophyletic, and both Collembola (i.e., Allacma) and ectognathian insects evolved independently from crustacean-like arthropods. However, the strongly supported, but evidently untenable, relationship of Lithobius and Strongylocentrotus argues for a cautious interpretation of the eIF2
tree. Combined analyses of eIF2
and other sequence data will deliver more soundly based phylogenies.
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Cladistic Patterns of Specific Introns
We further examined whether specific intron positions of eIF2 might be phylogenetically informative. Several cases of successive losses and gains of only slightly different intron positions were documented (fig. 5), resulting in a nested distribution of the evolutionary newer introns. Such nearby introns cannot coexist in one gene structure for two reasons. First, exon sizes smaller than 50 nt are seldom and functionally detrimental (see above) (Hwang and Cohen 1997; Carlo, Sierra, and Berget 2000). Second, co-occurring processes of intron gain and loss were suggested to be driven by balance between additional mutational load of intron-containing alleles and selective pressure for an efficient mechanism of NMD, which is provided by a sufficiently tight, overdispersed distribution of introns; that is, exon sizes are more uniform than expected under random insertion (Lynch and Kewalramani 2003). Therefore, such intron changes represent reliable synapomorphic character states.
The following cases of nested intron distributions are particularly informative. First, intron 212-1 was found in several species of animals, fungi, and protists. The nearby intron position 212-0 was identified only in angiosperm plants and may demarcate a monophyletic group of plants, because the intron 212-1 had been very likely lost before an intron 212-0 was evolved. Second, intron 127-2 was detected in protists, in some animals, and in one fungi (the basidiomycet Cryptococcus). A nesting intron position, only 9 nt away, is 130-2, which was exclusively found in all five analyzed Pezizomycotina species. In this case, we assume that ancient Ascomyceta eIF2 genes were intronless, because all other analyzed ascomycet species (Saccharomyces, Candida, Kluyveromyces, Eremothecium, and Schizosaccharomyces) have completely intronless eIF2
genes, and all analyzed Pezizomycotina species contain introns, specific only for this taxa (130-2 and 460-2). Interestingly, the establishment of spliceosomal introns in former intronless genes were already reported from Pezizomycotina species (Bhattacharya et al. 2000, and references therein). Third, nearly all analyzed eIF2
genes of Coleomata contain the intron 159-1, with exception of the analyzed Coleoptera, Lepidoptera, and Diptera, which instead contain the taxa-specific intron 160-1 (Anopheles is intronless in this region). In contrast, both the aphid Aphis sambuci and the bee Apis mellifera have the plesiomorphic intron 159-1 as deuterostomes and remotely related arthropods. Therefore, we propose a nested monophyletic taxa, a group including Diptera, Lepidoptera, and Coleoptera but not Hymenoptera (fig. 5). This novel taxa is additionally supported by the intron 295-0 of Clytus arietis (Coleoptera) and Bombyx mori (Lepidoptera), which is nested in distribution of the nearby intron 289-0 (found in platworms, deuterostomes, and several arthropods, including Apis mellifera). Our novel grouping contradicts commonly supported insect phylogeny, which considered the Coleoptera as outgroup to Hymenoptera+Diptera (Wheeler et al. 2001, and references therein). However, Diptera+Lepidoptera+Coleoptera excluding Hymenoptera was at least supported by Ross (1965) based on morphological arguments.
The taxonomic distribution of the intron positions 159-1 and 160-1 let us to assume that intron 160-1 might have originated by sliding of the 159-1 intron. Thus, we compared the 5' and 3' splice regions of both introns (fig. 6). We found a significant conservation of some nucleotide positions in the 3' splice sites of both introns, which argues, indeed, for the possibility of intron sliding involving at least three single-nucleotide substitutions. These substitutions do not necessarily need to occur contemporarily because of the implicated one-codon shift of both splice sites.
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Intron Positioning in eIF2 Reveal Insights in Phylogenetics and in Modes of Intron Evolution
Our results give support to the following hypothesis about the evolutionary history of eIF2 in eukaryotes. Intronless eIF2
genes were probably inherited by unicellular eukaryotes. First introns might have been acquired during early evolution and passed on to protists, plants, fungi, and animals. Other introns were gained significantly later. The value of these late introns for phylogenetics depends critically on their evolutionary polarization through nearby older introns, which have to be lost before the insertion of the novel introns, except in case of intron sliding, where intron loss and gain occurs simultaneously. As demonstrated by the ultrashort eIF2
exons found in fungal species (16 or 23 nt long, respectively), the detection of phylogenetically nested, synapomorphic introns can be complicated by unusual splicing features. Therefore, the reliability of this intron age classification depends critically (1) on the strong conservation of gene sequence, which makes the secure differentiation from nearby intron positions possible, and (2) on the sampling of gene structures, which has to be as tight as possible. The use of intron positioning for maximum-parsimony tree reconstruction analysis of remote related eukaryotes was relatively successful, which argues for a high phylogenetic information content of intron positions. This analysis is substantially backed by tree reconstruction based on protein sequences. For instance, both amino acid sequences and intron positions argue against an Ecdysozoa group containing both arthropods and nematodes. The strongly persistent intron 81-1 adds evidence to the Pancrustacea hypothesis. Two parallel, nested intron distributions delivered evidence for a novel monophyletic taxa, a Diptera+Lepidoptera+Coleoptera clade excluding Hymenoptera (Fig. 5). Finally, we suggest that further analyses of strongly conserved gene structures will continue to improve the knowledge of both intron evolution and higher-level phylogenetics.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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References |
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