- Research article
- Open Access
The alveolate translation initiation factor 4E family reveals a custom toolkit for translational control in core dinoflagellates
© Jones et al.; licensee BioMed Central. 2015
- Received: 29 August 2014
- Accepted: 29 January 2015
- Published: 10 February 2015
Dinoflagellates are eukaryotes with unusual cell biology and appear to rely on translational rather than transcriptional control of gene expression. The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in regulating gene expression because eIF4E binding to the mRNA cap is a control point for translation. eIF4E is part of an extended, eukaryote-specific family with different members having specific functions, based on studies of model organisms. Dinoflagellate eIF4E diversity could provide a mechanism for dinoflagellates to regulate gene expression in a post-transcriptional manner. Accordingly, eIF4E family members from eleven core dinoflagellate transcriptomes were surveyed to determine the diversity and phylogeny of the eIF4E family in dinoflagellates and related lineages including apicomplexans, ciliates and heterokonts.
The survey uncovered eight to fifteen (on average eleven) different eIF4E family members in each core dinoflagellate species. The eIF4E family members from heterokonts and dinoflagellates segregated into three clades, suggesting at least three eIF4E cognates were present in their common ancestor. However, these three clades are distinct from the three previously described eIF4E classes, reflecting diverse approaches to a central eukaryotic function. Heterokonts contain four clades, ciliates two and apicomplexans only a single recognizable eIF4E clade. In the core dinoflagellates, the three clades were further divided into nine sub-clades based on the phylogenetic analysis and species representation. Six of the sub-clades included at least one member from all eleven core dinoflagellate species, suggesting duplication in their shared ancestor. Conservation within sub-clades varied, suggesting different selection pressures.
Phylogenetic analysis of eIF4E in core dinoflagellates revealed complex layering of duplication and conservation when compared to other eukaryotes. Our results suggest that the diverse eIF4E family in core dinoflagellates may provide a toolkit to enable selective translation as a strategy for controlling gene expression in these enigmatic eukaryotes.
- Translation initiation
Dinoflagellates are unusual eukaryotic organisms well known for bloom formation in coastal waters , making toxins that bioaccumulate in the food chain , producing bioluminescence , and as coral symbionts. Dinoflagellate nuclei differ from those of other eukaryotes, exhibiting large birefringent chromosomes that lack typical eukaryotic nucleosomal DNA packaging. These chromosomes remain condensed during interphase and in many cases throughout the entire cell cycle . Dinoflagellate genomes can also be very large, up to hundreds of picograms of DNA per nucleus, making comprehensive genomic sequencing very difficult [5-7]. In addition, many dinoflagellate genes are duplicated with between tens to thousands of copies found for genes such as the peridinin chlorophyll protein, proliferating cell nuclear antigen, RuBisCO, light harvesting protein, luciferin binding protein, and actin [8-18]. The unusual features of the dinoflagellate nucleus are also reflected by atypical transcription .
In general, dinoflagellate gene expression appears to be controlled at the level of translation rather than transcription. For example, bioluminescence in dinoflagellates is strictly diurnally regulated, with little bioluminescence during the day and a peak during the night. Luciferin binding protein is made primarily at night, but transcript levels for the protein remained unchanged over a diel cycle . This example of translational regulation has been validated and expanded by numerous studies of heat shock, nutrient stress, salinity and other conditions using microarrays and high-throughput sequencing [21-26]. These studies have demonstrated a nearly constant transcriptome profile during stress with only small changes in transcript levels .
Given that dinoflagellates show little transcriptional control of gene expression, we looked for innovations in translation initiation unique to dinoflagellates that might enable specific translational control. Furthermore, dinoflagellate mRNA cap structures derive from the trans-splicing of a 22-nucleotide fragment from an independently transcribed molecule, the spliced leader RNA [28,29], which has a unique cap structure . In view of this, we have placed emphasis on a key step in mRNA recruitment, the binding of eukaryotic translation initiation factor 4E (eIF4E) to the modified base at the 5′ terminus of the mature mRNA (mRNA cap).
eIF4E is part of an extended gene family found exclusively in eukaryotes. Throughout the eukaryotic domain, a series of eIF4E gene duplications has led to the generation of a family of proteins with multiple structural classes and in some cases subclasses within a given organism [30,33,35]. Although the family is named for the translation initiation factor, not all members of the gene family function as translation factors [36,37]. This family is comprised of proteins predicted to have the novel fold. One or more eIF4E cognates bind the 5′- cap structure of mRNA, a key step in mRNA recruitment to the ribosome. Other eIF4E family members interact with specific mRNAs or proteins to regulate translation of those mRNAs rather than participate in global translation initiation [38-41]. Additional roles undertaken by eIF4E family members include nuclear transport, sequestration and turnover of mRNA [34-36]. Furthermore, some family members regulate translation of specific mRNAs rather than participate in global translation initiation. The eIF4E family can be seen as a toolkit for post-transcriptional regulation of gene expression .
It has been noted that the nomenclature for eIF4E family members has evolved with confusion . Eukaryotes have been reported to have at least one and up to eight members of the eIF4E family [33,35]. One proposed classification has divided the different members of the eIF4E family from multicellular organisms into structural classes; Class I, Class II, and Class III . Phylogenies of the eIF4E family are poorly resolved, but support monophyly of metazoan Classes I-III. Thus, while the structural classes define homologous, monophyletic clades within metazoans, across eukaryotes the evolutionary history of the family remains obscure .
The gene census and major clades for the heterokont and alveolate eIF4E family
The core dinoflagellate eIF4E family contains between eight and fifteen members per species with a total of 126 from eleven species (Additional file 1). In contrast, the members of the dinoflagellate lineage outside of the core dinoflagellates, Perkinsus marinus and Amoebophrya contain seven and three members per species, respectively. Of the sequences available for the other alveolate groups investigated (15 apicomplexans, 6 ciliates); apicomplexans have one or two family members per species, and ciliates have one to four. All of the ciliate sequences within eIF4E-1 and several within eIF4E-2 showed biased amino acid composition when compared with the remaining sequences, and ciliates consistently formed the longest branches in the phylogeny (Additional file 2) . The outgroup heterokonts (14 species) contain between one and six eIF4E family members per species.
Core dinoflagellate eIF4E-1 sub-clades
Core dinoflagellate eIF4E-1 contains four distinct monophyletic groups, each with at least one representative from each of the eleven core dinoflagellate species, which we have termed eIF4E-1a through -1d. Although bootstrap support for these groups was low when analyzed with the other two major eIF4E clades and outgroups, a re-analysis of eIF4E-1 containing only the core dinoflagellates produced a tree similar to that of the broader taxon sampling (Figure 3), but the sub-sampled tree had increased bootstrap support. Most notable was increased support for eIF4E-1d (100% from 36% bootstrap) and support for the combination of eIF4E-1a and -1b to 56%. Meanwhile, support remained high at 100% for eIF4E-1c.
There were different patterns of amino acid conservation between eIF4E-1a through -1d. eIF4E-1a was characterized by short branch lengths and high sequence identity. Selecting Amphidinium carterae and Karlodinium veneficum as representative core dinoflagellate species, there was 86% pairwise amino acid identity between the most closely related eIF4E-1a sequences across the entire sequence. This is roughly equal to the identity between the three eIF4E-1a copies in K. veneficum (83–87% identity). The eIF4E-1d is the next most conserved, with 70% identity between the two species. Divergence is higher in eIF4E-1b and -1c, with a pairwise identity between the same two species of 60% and 53%, respectively. We find asymmetric patterns of duplication across eIF4E-1a through -1d among core dinoflagellates. The rank order of number of copies per species was eIF4E-1a > −1d > −1b > −1c with eIF4E-1c having a single copy per species (Figure 3 inset). This is the same rank order that corresponds, from highest to lowest, to the pairwise similarity within each sub-clade.
Core dinoflagellate eIF4E-2 sub-clades
Within core dinoflagellate clade 2 eIF4E, two sub-clades were found, eIF4E-2a and -2b. eIF4E-2a was found in all eleven species, with eIF4E-2b being found in only eight species. eIF4E-2a is more conserved than eIF4E-2b based on pairwise comparisons and branch lengths. In contrast to eIF4E-1, there is higher bootstrap support for both eIF4E-2a and -2b (69% for eIF4E-2a and 100% for eIF4E- 2b). There is a large amino acid extension at the carboxy terminus of eIF4E-2a ranging from 200 – 270 amino acids compared to eIF4E-2b, a region not used in the phylogenetic analysis. This region was difficult to align between species and contains simple amino acid repeats of one proline followed by two to four alanines.
In three apicomplexan genera, Toxoplasma, Neospora and Plasmodium, eIF4E sequences that segregate with clade 2 eIF4E were found in initial phylogenies and searches. These sequences are very divergent, with long-branch lengths. Candidate homologs are absent from other apicomplexan groups in BLAST searches when either T. gondii or P. falciparum were used as a query. After initial phylogenies, these sequences were excluded from phylogenetic trees due to long-branch lengths (the GenBank gi numbers are reported in Additional file 1).
Core dinoflagellate eIF4E-3 sub-clades
Only the core dinoflagellates and heterokonts contain clade 3 eIF4E, and homologous sequences were absent in the apicomplexan and ciliate genome data based on BLAST searches and phylogeny. eIF4E-3 from heterokonts form two clades corresponding to the photosynthetic and non-photosynthetic heterokonts. Only one eIF4E-3 was found for each heterokont species examined. In contrast, eIF4E sequences from the core dinoflagellates separate into three monophyletic groups within clade 3 to give eIF4E-3a, −3b, and -3c. Like eIF4E-1 and eIF4E-2a, eIF4E-3a contains sequences from all eleven core-dinoflagellate species and is more conserved than eIF4E-3b and -3c. eIF4E-3b and -3c are poorly represented with just six and five species, respectively. eIF4E-3b sequences have an extended amino-terminus compared to eIF4E-3a, that includes a DNA alkylation, or dioxygenase, domain, a divergent character not captured by the phylogeny of the aligned core region. Additionally, eIF4E-3b contained a number of sequences with amino acid compositional bias when compared with the remaining members of the gene family (Additional file 2).
Comparison with eIF4E sequences from Perkinsus marinus and Amoebophrya
Within eIF4E-1, the two Amoebophrya strains, representing the syndineans each have two distinct copies, one forming an outgroup to the apicomplexan/core dinoflagellate clade, and a second copy nested near eIF4E-1a of core dinoflagellates. Neither of these placements is strongly supported by bootstrap values. For eIF4E-2, the syndineans have a single copy. The branching pattern and bootstrap support does not show a clear affinity with either eIF4E-2a or -2b, but based on sequence length, the Amoebophrya eIF4E-2 was more similar to eIF4E-2a. No eIF4E-3-like sequences were found in the genus Amoebophyra.
Perkinsus marinus falls within the dinoflagellate lineage based on molecular phylogenies, but outside of the core dinoflagellate and syndinean clades (Figure 2) [44,46,47,52]. The P. marinus eIF4E family contains a total of two eIF4E-1, one eIF4E-2 and four eIF4E-3 members. The eIF4E-1 and eIF4E-3 sequences form a single species-specific monophyletic group with bootstrap support of 92% and 90%, respectively. While the major clade affiliation was clear, the phylogeny did not suggest specific relationships between the P. marinus eIF4E and core dinoflagellate sub-clades.
The eIF4E phylogeny of apicomplexans, dinoflagellates and heterokonts was combined with the previously published analysis of Joshi et al.  and recapitulated the eight well-supported clades of that analysis. As was previously reported, the relationships between the eight clades is ambiguous with poor bootstrap support. Dinoflagellate, ciliate and apicomplexan (Alveolate) as well as heterokont eIF4E-1 were placed within the clade 8 of Joshi et al.  and the combined Alveolate and heterokont eIF4E-1 and −3 were recovered as monophyletic groups (Additional file 4). However, comparing the phylogeny in Figure 3 to the broader phylogeny, eIF4E-2 from heterokonts did not form a single clade and were placed outside of eIF4E-2 from dinoflagellates, albeit with poor support. Phylogenies that included the long-branch ciliates were even more difficult to interpret. The three major clades (eIF4E-1, −2, and −3) described here cannot be reliably matched to the three previously described eIF4E classes (Class I, Class II, and Class III) .
Structural predictions across the three dinoflagellate eIF4E clades
The alignment of the core dinoflagellate eIF4E family members used to construct the phylogenetic tree contains insertions, deletions, and residue changes at important functional positions that differentiate the clades and sub-clades (Figure 4). The eight conserved aromatic residues of murine eIF4E have been mapped onto the alignment of dinoflagellate eIF4E sequences (Figure 4) as points of reference. The eIF4E-1 contains extended sequences between W73 and W102, and W130 to W166 compared to eIF4E-2 and −3. The function of the extension is not known, but the different eIF4E-1 sub-clades show marked heterogeneity at this region. In the core dinoflagellate clade 1 and clade 2 eIF4E, there is a tyrosine substitution at the position equivalent to W56. Conserved W102 is maintained across eIF4E sequences from all dinoflagellate clades except eIF4E-3b. In this case, the hydrophobic tryptophan has been substituted for an arginine. E103, which also interacts with the mRNA cap, is found at equivalent positions in all three dinoflagellate clades of eIF4E [31,32]. However, in clade 2 eIF4E, there is a small deletion C-terminal to it, as well as the introduction of a proline residue; an indicator of possible kinks in the polypeptide and an alteration of its function. The tryptophans at positions 43 and 46 are conserved, as is W166, which is involved in recognition of the methyl group on m7GTP.
In core dinoflagellate eIF4E-3 sequences, R112 is conserved, but there is a conservative substitution of histidine in eIF4E-1 and a cysteine substitution in eIF4E-2. In all three dinoflagellate clades, R157 may or may not be conserved, because the alignment of this residue is ambiguous near the insertion between W130 and W166 of dinoflagellate eIF4E-1 (Figure 4). eIF4E-3 contains the conserved K162 and in eIF4E-1 there is an arginine substitution, but this site is a hydrophobic valine or alanine in eIF4E-2.
In eIF4E family members capable of binding eIF4G, the consensus sequence of the recognition motif is S/TVxxFW ending at W73 [31,32,56-58]. In the core dinoflagellate eIF4E sequences, similar motifs are conserved. Although eIF4E-1 and −3 have a tryptophan at W73, eIF4E-2 has tyrosine, phenyalanine or leucine. In addition, there are subtle variations between the different eIF4E-1 sub-clades in the consensus sequence of the binding domain. The region can be polar (TVQeFW), as in eIF4E-1a and -1b, acidic (TVEEFW) as in eIF4E-1c, or basic (TVKgFW), as in eIF4E-1d. Overall, based on primary sequence alignment and structural predictions, the members of the core dinoflagellate eIF4E family are diverse at sites known to be functionally important in mouse and other model organisms. Hence, functional analysis will be necessary to determine the roles of different family members.
Evolutionary patterns in the eIF4E family
The combination of relatively dense taxon sampling and known relationships has allowed us to make clear comparisons of the eIF4E genealogy to the organismal relationships, revealing both the relative timing and degree of duplication within core dinoflagellates. Prior to diversification of heterokonts and alveolates the eIF4E gene family contained at least three clades. These clades were retained in heterokonts and dinoflagellates, but apicomplexans and ciliates have apparently lost eIF4E-2 and −3 (Figure 3). Other shared eIF4E family members may have been lost from either dinoflagellates or heterokonts or both. The large sequence divergence between the three clades implies different functions, so that the common ancestor is likely to have used the gene family in three different roles. However, in the core dinoflagellates the gene family has expanded mostly via relatively recent, lineage specific duplications, into nine distinct eIF4E sub-clades. Additional duplications within sub-clades have been found, so that some species have multiple subtly different copies. The multiple eIF4E family members have the potential to create a customized toolkit for regulating gene expression as in plants and metazoans. In contrast, heterokonts have only four and apicomplexans and ciliates only one or two eIF4E family members, suggesting that different lineages have placed different reliance on, and approaches to, translational control of gene expression.
Matching eIF4E structural classes to clades
Based on phylogenetic analysis the three eIF4E clades described here cannot be mapped to the three previously defined eIF4E structural classes in animals and plants (Additional file 4) [30,33]. The broad eIF4E phylogeny is poorly resolved and the underlying organismal relationships between metazoa, plants, heterokonts and alveolates are uncertain (Additional file 4) [30,33]. For example, based on the phylogeny, the dinoflagellate eIF4E-1 defined here has no obvious candidate ortholog in plants or metazoans. The eIF4E-1 sequences from alveolates and heterokonts contain an amino acid insertion (Figure 3) between W73 and W102 (numbering equivalent to murine eIF4E), a feature not seen in any plant or metazoan eIF4E family member (Figure 4). Alternately, plant and metazoan Class II eIF4E family members are defined in part by a tyrosine, phenylalanine or leucine at the position equivalent to murine W43. By this definition, no Class II eIF4E sequences have been found in dinoflagellates or ciliates. However, heterokonts do contain eIF4E family members defined as metazoan Class II based on this diagnostic character. The non-reciprocal mapping of clades to classes suggests diverse approaches to eIF4E functions in eukaryotes with loss and duplication occurring multiple times independently within plants, fungi, metazoans, alveolates and heterokonts.
Different functions are likely among the dinoflagellate eIF4E family members
For the few gene families that have been studied in core dinoflagellates, a general pattern of highly duplicated but conserved genes in tandem arrays has been seen for actin, proliferating cell nuclear antigen, and the peridinin chlorophyll protein [9,11,12,14,15]. The different sequences typically have few amino acid differences and many synonymous nucleotide substitutions, suggesting little functional difference between the resultant proteins. Several other gene families, such as RuBsiCO, luciferase, and the light harvesting complex protein are polyproteins, with tandemly repeated protein units encoded on a single mRNA [10,13,59]. Our work shows that the eIF4E family contrasts with these other examples because the divergences are seen at critical amino acids suggesting the family members are functionally distinct. The broad species representation we used has allowed inferences about the relative timing of duplication and species divergence to be made and suggests that the different sub-clades within eIF4E-1 were likely present in the common ancestor of core dinoflagellates. Differences in duplication and divergence within each clade and sub-clade may reflect a form of genomic dose effect, such that the more essential eIF4E forms are also the more duplicated and possibly more specialized. Thus, although the functionalities present in the common ancestor of alveolates and heterokonts are likely to be retained in the three clades, the sub-clades present in the core dinoflagellates are likely to play distinct roles in the cell and correlate with an increased reliance on post-transcriptional control.
Dinoflagellate clade 1 eIF4E is likely to contain at least one translational initiation factor
Of the three major clades in the phylogeny shown here (Figure 3), eIF4E-1 was the only clade that included sequences from every species surveyed. Within core dinoflagellates, eIF4E-1 stands out as the most duplicated, ranging from 5 to 9 copies per species and is also the most conserved. Within eIF4E-1a, only 15% of amino acid sites were found to be variable across the entire coding sequence, and the changes represent functionally conservative substitutions, i.e. high similarity or many positives in pairwise comparisons. Although all plant, fungal, and metazoan translation initiation factors are members of Class I, not all Class I eIF4E members are initiation factors [34,60]. By analogy, not all dinoflagellate clade 1 eIF4E family members may be functional translational initiation factors. Based on conservation and duplication, we predict that the primary workhorse translation initiation factor will be found within eIF4E-1, as this appears to be the only eIF4E present in apicomplexans. Also, if the theory of dose compensation holds true, the rank order of number of copies per species; i.e. eIF4E-1a > d > b > c, would suggest that eIF4E-1a functions as the primary translation factor.
In looking for different roles of the other sub-clades in the regulation of gene expression, a variety of possibilities exist. Simplistically, should all the eIF4E-1 function as translation initiation factors, eIF4E-1a could be involved in recruitment of the most commonly translated mRNAs, followed by -1d, −1b, and -1c. Examples of this can be seen in certain metazoan Class I eIF4Es. For instance, in Caenorhabditis elegans, functional differences are found within the different members of Class I eIF4E. At specific developmental stages, C. elegans trans-splices sub-sets of mRNAs with a spliced leader containing a trimethyl cap structure . In C. elegans, m7GTP and trimethyl mRNA caps are bound by different Class I eIF4E family members, IFE3 versus IFE1, 2 and 5, respectively [62-64]. Although all core dinoflagellate mRNAs are presumed to be trans-spliced, it seems that the first nucleotide after the methylated base can be variable, opening up the possibility that the four eIF4E-1 sub-clades could recruit mRNAs with different cap structures [65-67].
The conservation found in clade 1 eIF4E does not extend to the eIF4G binding domain, in which there are amino acid substitutions within each sub-clade that are likely to have strong affinities for different binding partners. The eIF4G binding domain is also the site of interaction of a range of eIF4E binding proteins that use the same YXXXXLφ sequence to competitively bind eIF4E, preventing eIF4E–eIF4G interactions and inhibiting cap-dependent translation and/or targeting mRNAs to specific locations [34,60]. The variable sequence of the eIF4G binding domain represents a strategic point for selection of mRNAs. Different eIF4E and eIF4G combinations could favor the translation of different subsets of mRNA thus providing a toolkit for regulation of gene expression post-transcriptionally as has been proposed in Drosophila melanogaster [41,68] and Leishmania [69,70].
Dinoflagellate eIF4E-2 has an alternate function in the toolkit
Core dinoflagellate eIF4E-2 have substitutions at two positively charged residues known to be involved in cap binding, R112 and K162 (Figures 1 and 4). In eIF4E-1, a histidine is conserved in all sub-clades (Figure 4), but in eIF4E-2, cysteine is substituted at position 112. In eIF4E-1 at K162 a conserved arginine is present in all sub-clades, but this has been substituted by a valine in eIF4E-2. These substitutions could inhibit interaction with the negatively charged phosphate groups linking the cap to the mRNA. Furthermore, there is a two amino acid deletion near the conserved W102 involved in cap binding. In consideration of these differences, we predict that eIF4E-2 does not function well as a cap binding protein in vitro.
Differences in cap binding ability are reminiscent of the metazoan Class II and Class III eIF4Es that cannot compete in vitro with Class I eIF4E for mRNA and are not found associated with eIF4G in cells [38,39,43,71]. However, the affinity of metazoan Class II eIF4E, to bind mRNA 5′-caps is increased by interaction with a variety of partner proteins that, along with metazoan Class II eIF4E function to repress specific mRNA translation [38,39,43,71]. Similarly, metazoan Class III eIF4E specifically binds the m7G cap in the absence of an aromatic sandwich, using instead different spatial arrangements of residues to provide the necessary electrostatic and van der Waals contacts [43,71]. Although the eIF4E from Class III has a lower affinity than Class I eIF4E for mRNA caps in vitro, in cells nearly all of the endogenous mouse Class III eIF4E is in the cap-bound fraction suggesting that factors in the cell increase the affinity of Class III eIF4E for the cap [43,71]. Given these examples in metazoa, and the key substitutions in eIF4E-2 cap binding residues, dinoflagellate eIF4E-2 alone are unlikely to bind cap, but may bind in the presence of interacting partner proteins.
Clade 2 eIF4E is also unique in that the degree of duplication is roughly comparable within core dinoflagellates and heterokonts. Heterokonts contain two different cognates within eIF4E-2, but only one was allied with the dinoflagellates, while the other is allied with Class II eIF4E from metazoans and plants. Whether or not the placement of heterokont sequences with Class II sequences is convergent evolution cannot be stated from these data. However, retention of eIF4E-2 in heterokonts and dinoflagellates (both core dinoflagellates and syndineans) implies a conserved function that does not rely on traditional cap binding. Also the extension at the carboxy terminus of eIF4E-2b indicates eIF4E-2b and possibly eIF4E-2 interact with binding partners that are not associated with eIF4E-1 or −3. The absence of eIF4E-2 in several of the alveolate lineages and differences in duplication when comparing dinoflagellates and heterokonts makes it likely that eIF4E-2 performs a non-essential regulatory role which has become more specialized within the dinoflagellates.
Core dinoflagellate eIF4E-3 have many attributes in common with the metazoan Class I eIF4E
Unlike core dinoflagellate eIF4E-1 and eIF4E-2, eIF4E-3a and -3c have a tryptophan at position 56 and show complete conservation of the amino acids important in charge neutralization of the phosphate bridge of the mRNA cap. They do not have the insertions uniquely characteristic of eIF4E-1. In addition, the eIF4G interacting motif more closely resembles that of the metazoan Class I eIF4E. However, for multiple considerations, eIF4E-3 has not been considered a good candidate for the workhorse translation factor. These include the fact that eIF4E-3 is found only in the core dinoflagellates and heterokonts, and is absent in the apicomplexan and ciliate genome data. Additionally, only one eIF4E-3 is found for any heterokont species examined with the two clades corresponding to the photosynthetic and non-photosynthetic heterokonts. Furthermore, only one eIF4E-3 sub-clade, eIF4E-3a, is represented in all eleven core-dinoflagellate species with eIF4E-3b and eIF4E-3c represented in just six and five species, respectively. Moreover, the fusion of the alkylation or dioxygenase domain on the N-terminus of eIF4E-3b is remarkable and unprecedented, making hypotheses about its function unrealistic without further experiments. This does not mean, however, that eIF4E-3 does not contain a translation factor.
There are certainly differences in the conserved cap binding pocket between members of eIF4E-1 and −3 and it is not clear that individual spliced leader mRNA in dinoflagellates possess an identical cap [65-67]. Differences in mRNA caps could allow specific translation of genes involved in stress response or other unique pathways that would not be constitutively expressed. Conversely, expression of members of eIF4E-3 could be situational; only occurring during specific developmental stages and not detectable at other times.
Dinoflagellate genomes contain an eIF4E family that demonstrates a greater diversity and degree of duplication than has been seen in other eukaryotes . The general model for metazoan eIF4E family members is to have different cellular roles related to post-transcriptional regulation of gene expression. These different roles are supported by phylogeny and specific amino acid changes mapped onto the protein structure . Likewise, dinoflagellate eIF4E family members are expected to have distinct functions based on this phylogeny, with their unique diversity allowing for an increased dependence on the translational control of gene expression. Only through structural and functional studies can we confirm these predictions made from the phylogeny.
Data from Illumina RNA-seq libraries were used for eleven core dinoflagellate species and two syndineans. Six of these, Akashiwo sanguinea, Amphidinium carterae, Gyrodinium instriatum, Karlodinium veneficum, Polarella glacialis, and Prorocentrum minimum were sequenced with 100 base paired-end reads with 30 to 100 million reads collected per species and assembled with Trinity [44,72] (Additional file 1). The two syndineans, Amoebophrya parasites specific for Akashiwo sanguinea and Karlodinium veneficum, respectively, were co-cultured and sequenced with their hosts as previously described . Parasite sequences were deconvoluted from host using either using AT bias for the parasite from Karlodinium veneficum, or by phylogenetic analysis for Amoebophrya sp. ex Akashiwo sanguinea as done previously . In addition, Moore Foundation Illumina datasets were downloaded for five core dinoflagellates. These consisted of 50 base paired-end reads for Alexandrium tamarense, Karenia brevis, Lingulodinium polyedrum, and Scrippsiella trochoidea. The sequences were downloaded from the Community cyberinfrastructure for Advanced Microbial Ecology Research and Analysis, CAMERA, database (http://camera.crbs.ucsd.edu/mmetsp/) and assembled using CLC Genomics (Qiagen, Boston, MA). Transcriptome data for Symbiodinium sp. B1 were downloaded from http://marinegenomics.oist.jp/genomes/downloads?project_id=21 .
In addition to the eleven core dinoflagellates and two syndinians, the genome sequences from Perkinsus marinus and twelve apicomplexans [see Additional file 1] were also used. Data from GenBank’s reference sequence (ref_seq) protein or non-redundant nucleotide database were downloaded and formatted into blast databases for each species. In total, sequences from forty-nine different species were used in this study [see Additional file 1].
eIF4E sequence retrieval and annotation
The blastx or blastp programs were used with an e-value cut off of 1E−10 and a suite of eIF4E query sequences . For dinoflagellates, the set of eIF4E sequences from two representative species, Amphidinium carterae and Karlodinium veneficum were used as queries. For heterokonts, eIF4E sequences from Thalassiosira pseudonana and Phaeodactylium pseudonana were used as queries. For apicomplexans, Toxoplasma gondii and Plasmodium vivax were used as queries. Such searches consistently returned pairwise alignments of >130 aligned amino acids. The resulting eIF4E sequences were aligned using the Clustal Omega program with the full iteration option .
Sequences from the representative species within each clade identified in the phylogeny were used as queries in subsequent blast searches to improve representation. This iterative process was repeated until no novel eIF4E family members were found. For a few sequences, manual assembly of scaffolded sequences was required to obtain complete coding regions. In such cases, promising blast hits (e-values < 1E−10) with short alignments were examined using read mapping to identify and then manually assemble fragmented gene copies. This was done using Bowtie2 read mapping of reads to contigs, followed by visualization using the Integrative Genomics Viewer (IGV) to identify read mates, and finally by assembly using short overlap consensus in Sequencher (Gene Codes, Ann Arbor, MI) [76,77]. A putative heterokont contaminant was found in the sequence data from Alexandrium tamarense and removed from further analyses (Additional file 4).
The alignments were trimmed to the eIF4E core region from five amino acids upstream of W43 to ten amino acids downstream of W166 numbered by murine eIF4E [PDB:1L8B] sequence positions . The sequence alignment was submitted to TreeBase with accession number [S16938]. Phylogenetic analysis was performed using RAxML with 1000 bootstrap replicates. The LG amino acid substitution matrix with gamma rate correction was selected based on hierarchical likelihood ratio tests of the optimal tree calculated using the JTT amino acid substitution matrix (the PROTGAMMAJTT model in RAxML) . Using the amino acid frequencies from the LG matrix resulted in better likelihood scores than the empirical amino acid frequencies in the alignment, and similar results were seen with the WAG and JTT matrices. All the analyses assumed a single stationary model across the tree. An additional phylogeny was constructed using the supplemental data from Joshi et al. and adding in the sequences from the present study with 100 bootstrap replicates, although in this case the JTT model was used . For this combined analysis, the ciliates were excluded due to long-branch lengths.
Amino acid bias was calculated using Tree-Puzzle which uses a chi-squared test to compare the amino acid composition of each alignment row with the empirical frequencies calculated from the alignment . The amino acid bias for specific clades was further explored using the composition profiler tool .
Sequence logos were created using WebLogo  for each core dinoflagellate clade by trimming outgroup taxa and creating clade specific alignments. The amino acid sequences were aligned, and the secondary and tertiary structures predicted using an online Protein Homology/analogY Recognition Engine (PHYRE) , with [PDB:1L8B] as a reference. The resulting PDB file was then displayed with Visual Molecular Dynamics software (http://www.ks.uiuc.edu/Research/vmd/) .
Availability of supporting data
Alignments used in this study were deposited in TreeBase with accession # S16938, see http://purl.org/phylo/treebase/phylows/study/TB2:S16938.
This work was funded by grants from Oceans & Human Health NIH R01ES021949-01/NSF OCE1313888 to R.J. and A.R.P. Many thanks to Charles F. Delwiche and the Dinoflagellate Assembling the Tree of Life Grant (DEB-0629624). Thanks also to the Moore Foundation and CAMERA for additional datasets. This is contribution # 4989 of the University of Maryland Center for Environmental Science and # 15-143 of the Institute of Marine and Environmental Technology.
- Anderson DM, Alpermann TJ, Cembella AD, Collos Y, Masseret E, Montresor M. The globally distributed genus Alexandrium: multifaceted roles in marine ecosystems and impacts on human health. Harmful Algae. 2012;14:10–35.View ArticlePubMed CentralPubMedGoogle Scholar
- Holmes MJ, Brust A, Lewis RJ. Dinoflagellate Toxins: An overview. In: Botana LM, editor. Seafood and Freshwater Toxins: Pharmacology, physiology and detection, Third edition. Boca Raton, FL: CRC Press; 2014.Google Scholar
- Kelly MG, Tett P: Bioluminescence in the ocean. In: Bioluminescence in action. Academic Press; 1978.Google Scholar
- Spector DL. Dinoflagellate Nuclei. In: Spector DL, editor. Dinoflagellates. London: Academic Press; 1984. p. 107–47.View ArticleGoogle Scholar
- Lin S. Genomic understanding of dinoflagellates. London. Res Microbiol. 2011;162(6):55–569.View ArticleGoogle Scholar
- Wisecaver JH, Hackett JD. Dinoflagellate genome evolution. Annu Rev Microbiol. 2011;65:369–87.View ArticlePubMedGoogle Scholar
- Shoguchi E, Shinzato C, Kawashima T, Gyoja F, Mungpakdee S, Koyanagi R, et al. Draft Assembly of the Symbiodinium minutum Nuclear Genome Reveals Dinoflagellate Gene Structure. Curr Biol. 2013;23(15):1399–408.View ArticlePubMedGoogle Scholar
- Bachvaroff TR, Concepcion GT, Rogers CR, Delwiche CF. Dinoflagellate EST data indicate massive transfer of chloroplast genes to the nucleus. Protist. 2004;55(1):65–78.View ArticleGoogle Scholar
- Bachvaroff TR, Place AR. From stop to start: tandem gene arrangement, copy number and trans-splicing sites in the dinoflagellate Amphidinium carterae. PLoS One. 2008;3(8):e2929.View ArticlePubMed CentralPubMedGoogle Scholar
- Hiller RG, Wrench PM, Sharples FP. The light harvesting chlorophyll a-c-binding protein of dinoflagellates: a putative polyprotein. FEBS Lett. 1995;363:175–8.View ArticlePubMedGoogle Scholar
- Kim S, Bachvaroff TR, Handy SM, Delwiche CF. Dynamics of actin evolution in dinoflagellates. Mol Biol Evol. 2011;28:1469–80.View ArticlePubMedGoogle Scholar
- Reichman J, Wilcox T, Vize P. PCP Gene Family in Symbiodinium from Hippopus hippopus: Low level of concerted evolution, isoform diversity and spectral tuning of chromophores. Mol Biol Evol. 2003;20(12):2143–54.View ArticlePubMedGoogle Scholar
- Rowan R, Whitney SM, Fowler A, Yellowlees D. Rubisco in marine symbiotic dinoflagellates: form II enzymes in eukaryotic oxygenic phototrophs encoded by a nuclear multigene family. Plant Cell. 1996;8:539–53.View ArticlePubMed CentralPubMedGoogle Scholar
- Zhang H, Hou Y, Lin S. Isolation and characterization of proliferating cell nuclear antigen from the dinoflagellate Pfeisteria piscicida. J Eukaryot Microbiol. 2006;53(2):142–50.View ArticlePubMedGoogle Scholar
- Le QH, Markovic P, Hastings JW, Jovine RV, Morse D. Structure and organization of the peridinin-chlorophyll a-binding protein gene in Gonyaulax polyedra. Mol Gen Genet. 1997;255(6):595–604.View ArticlePubMedGoogle Scholar
- Valiadi M, Iglesias-Rodriguez MD, Amorim A. Distribution and genetic diversity of the luciferase gene within marine dinoflagellates. J Phycol. 2012;48:826–36.View ArticleGoogle Scholar
- Liu L, Hastings J. Novel and rapidly diverging intergenic sequences between tandem repeats of the luciferase genes in seven dinoflagellate species. J Phycol. 2005;42(1):96–103.View ArticleGoogle Scholar
- Liu L, Wilson T, Hastings JW. Molecular evolution of dinoflagellate luciferases, enzymes with three catalytic domains in a single polypeptide. Proc Natl Acad Sci U S A. 2004;101(47):16555–60.View ArticlePubMed CentralPubMedGoogle Scholar
- Guillebault D, Sasorith S, Derelle E, Wurtz JM, Lozano JC, Bingham S, et al. A new class of transcription initiation factors, intermediate between TATA box-binding proteins (TBPs) and TBP-like factors (TLFs), is present in the marine unicellular organism, the dinoflagellate Crypthecodinium cohnii. J Biol Chem. 2002;277(43):40881–6.View ArticlePubMedGoogle Scholar
- Morse D, Milos PM, Roux E, Hastings JW. Circadian regulation of bioluminescence in Gonyaulax involves translational control. Proc Natl Acad Sci U S A. 1989;86(1):172–6.View ArticlePubMed CentralPubMedGoogle Scholar
- Toulza E, Shin MS, Blanc G, Audic S, Laabir M, Collos Y, et al. Gene expression in proliferating cells of the dinoflagellate Alexandrium catenella (Dinophyceae). Appl Environ Microbiol. 2010;76(13):4521–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Lidie KB, Ryan JC, Barbier M, Van Dolah FM. Gene expression in Florida red tide dinoflagellate Karenia brevis: Analysis of an expressed sequence tag library and development of a DNA microarray. Mar Biotechnol (NY). 2005;7:481–93.View ArticleGoogle Scholar
- Morey JS, Monroe EA, Kinney AL, Beal M, Johnson JG, Hitchcock GL, et al. Transcriptomic response of the red tide dinoflagellate, Karenia brevis, to nitrogen and phosphorus depletion and addition. BMC Genomics. 2011;12:346.View ArticlePubMed CentralPubMedGoogle Scholar
- Lowe CD, Mello LV, Samatar N, Martin LE, Montagnes DJ, Watts PC. The transcriptome of the novel dinoflagellate Oxyrrhis marina (Alveolata: Dinophyceae): response to salinity examined by 454 sequencing. BMC Genomics. 2011;12:519.View ArticlePubMed CentralPubMedGoogle Scholar
- Moustafa A, Evans AN, Kulis DM, Hackett JD, Erdner DL, Anderson DM, et al. Transcriptome profiling of a toxic dinoflagellate reveals a gene-rich protist and a potential impact on gene expression due to bacterial presence. PLoS One. 2010;5(3):e9688.View ArticlePubMed CentralPubMedGoogle Scholar
- Yang I, John U, Beszteri S, Glockner G, Krock B, Goesmann A, et al. Comparative gene expression in toxic versus non-toxic strains of the marine dinoflagellate Alexandrium minutum. BMC Genomics. 2010;11:248.View ArticlePubMed CentralPubMedGoogle Scholar
- Roy S, Morse D. Transcription and maturation of mRNA in dinoflagellates. Microorganisms. 2013;1:71–99.View ArticleGoogle Scholar
- Lidie KB, Van Dolah FM. Spliced leader RNA-mediated trans-splicing in a dinoflagellate, Karenia brevis. J Eukaryot Microbiol. 2007;54(5):427–35.View ArticlePubMedGoogle Scholar
- Zhang J, Hou Y, Miranda L, Campbell D, Sturm N, Gaasterland T, et al. Spliced leader RNA trans-splicing in dinoflagellates. Proc Natl Acad Sci. 2007;104(11):4618–23.View ArticlePubMed CentralPubMedGoogle Scholar
- Jagus R, Bachvaroff TR, Joshi B, Place AR. Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists. Comp Funct Genomics. 2012;2012(13):483–9.Google Scholar
- Marcotrigiano J, Gingras AC, Sonenberg N, Burley SK. Cocrystal structure of the messenger RNA 5′ cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell. 1997;89(6):951–61.View ArticlePubMedGoogle Scholar
- Matsuo H, Li H, McGuire AM, Fletcher CM, Gingras AC, Sonenberg N, et al. Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein. Nat Struct Biol. 1997;4(9):717–24.View ArticlePubMedGoogle Scholar
- Joshi B, Lee K, Maeder DL, Jagus R. Phylogenetic analysis of eIF4E-family members. BMC Evol Biol. 2005;5:48.View ArticlePubMed CentralPubMedGoogle Scholar
- Kamenska A, Simpson C, Standart N. eIF4E-binding proteins: new factors, new locations, new roles. Biochem Soc Trans. 2014;42(4):1238–45.View ArticlePubMedGoogle Scholar
- Hernandez G, Altmann M, Sierra JM, Urlaub H, Diez del Corral R, Schwartz P, et al. Functional analysis of seven genes encoding eight translation initiation factor 4E (eIF4E) isoforms in Drosophila. Mech Dev. 2005;122(4):529–43.View ArticlePubMedGoogle Scholar
- Aitken CE, Lorsch JR. A mechanistic overview of translation initiation in eukaryotes. Nat Struct Mol Biol. 2012;19(6):568–76.View ArticlePubMedGoogle Scholar
- Hinnebusch AG. Translational homeostasis via eIF4E and 4E-BP1. Mol Cell. 2012;46(6):717–9.View ArticlePubMedGoogle Scholar
- Cho PF, Poulin F, Cho-Park YA, Cho-Park IB, Chicoine JD, Lasko P, et al. A new paradigm for translational control: inhibition via 5′-3′ mRNA tethering by Bicoid and the eIF4E cognate 4EHP. Cell. 2005;121(3):411–23.View ArticlePubMedGoogle Scholar
- Morita M, Ler LW, Fabian MR, Siddiqui N, Mullin M, Henderson VC, et al. A novel 4EHP-GIGYF2 translational repressor complex is essential for mammalian development. Mol Cell Biol. 2012;32(17):3585–93.View ArticlePubMed CentralPubMedGoogle Scholar
- Villaescusa JC, Verrotti AC, Ferretti E, Farookhi R, Blasi F. Expression of Hox cofactor genes during mouse ovarian follicular development and oocyte maturation. Gene. 2004;330:1–7.View ArticlePubMedGoogle Scholar
- Hernandez G, Han H, Gandin V, Fabian L, Ferreira T, Zuberek J, et al. Eukaryotic initiation factor 4E-3 is essential for meiotic chromosome segregation, cytokinesis and male fertility in Drosophila. Development. 2012;139(17):3211–20.View ArticlePubMed CentralPubMedGoogle Scholar
- Tettweiler G, Kowanda M, Lasko P, Sonenberg N, Hernandez G. The Distribution of eIF4E-Family Members across Insecta. Comp Funct Genomics. 2012;2012:960420.View ArticlePubMed CentralPubMedGoogle Scholar
- Osborne MJ, Volpon L, Kornblatt JA, Culjkovic-Kraljacic B, Baguet A, Borden KL. eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition. Proc Natl Acad Sci U S A. 2013;110(10):3877–82.View ArticlePubMed CentralPubMedGoogle Scholar
- Bachvaroff TR, Gornik SG, Concepcion GT, Waller RF, Mendez GS, Lippmeier JC, et al. Dinoflagellate phylogeny revisited: Using ribosomal proteins to resolve deep branching dinoflagellate clades. Mol Phylogenet Evol. 2014;70:314–22.View ArticlePubMed CentralPubMedGoogle Scholar
- Hoppenrath M, Leander BS. Dinoflagellate phylogeny as inferred from heat shock protein 90 and ribosomal gene sequences. PLoS One. 2010;5(10):e13220.View ArticlePubMed CentralPubMedGoogle Scholar
- Goggin CL, Barker SC. Phylogenetic position of the genus Perkinsus (Protista, Apicomplexa) based on small subunit ribosomal RNA. Mol Biochem Parasitol. 1993;60(1):65–70.View ArticlePubMedGoogle Scholar
- Reece KS, Siddall ME, Burreson EM, Graves JE. Phylogenetic analysis of Perkinsus based on Actin Gene Sequences. J Parasitol. 1997;83(3):417–23.View ArticlePubMedGoogle Scholar
- Saldarriaga JF, McEwan ML, Fast NM, Taylor FJR, Keeling PJ. Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage. IJSEM. 2003;53:355–65.PubMedGoogle Scholar
- Gajadhar AA, Marquardt WC, Hall R, Gunderson J, Ariztia-Carmona EV, Sogin ML. Ribosomal RNA sequences of Sarcocystis muris, Theileria annulata and Crypthecodinium cohnii reveal evolutionary relationships among apicomplexans, dinoflagellates, and ciliates. Mol Biochem Parasitol. 1991;45:147–54.View ArticlePubMedGoogle Scholar
- Baldauf S. The deep roots of eukaryotes. Science. 2003;5626:1703–6.View ArticleGoogle Scholar
- Burki F, Shalchian-Tabrizi K, Minge M, Skjaevelenad A, Nikolaev S, Jakobsen KS, et al. Phylogenomics reshuffles the eukaryotic supergroups. PLoS One. 2007;2(8):e790.View ArticlePubMed CentralPubMedGoogle Scholar
- Bachvaroff TR, Handy SM, Place AR, Delwiche CF. Alveolate phylogeny inferred using concatenated ribosomal proteins. J Eukaryot Microbiol. 2011;58(3):223–33.View ArticlePubMedGoogle Scholar
- Guillou L, Viprey M, Chambouvet A, Welsh RM, Kirkham AR, Massana R, et al. Widespread occurrence and genetic diversity of marine parasitoids belonging to Syndiniales (Alveolata). Environ Microbiol. 2008;10:3349–65.View ArticlePubMedGoogle Scholar
- Coats DW, Kim S, Bachvaroff TR, Handy SM, Delwiche CF. Tintinnophagus acutus n.g., n. sp. (Phylum Dinoflagellata), an Ectoparasite of the Ciliate Tintinnopsis cylindrica Daday 1887, and Its Relationship to Duboscquodinium collini Grassse 1952. J Eukaryot Microbiol. 2010;57:468–82.View ArticlePubMedGoogle Scholar
- Bachvaroff TR, Handy SM, Place AR, Delwiche CF. Alveolate phylogeny using ribosomal proteins. J Eukar Microbiol. 2011;58(3):223–33.View ArticleGoogle Scholar
- Tomoo K, Shen X, Okabe K, Nozoe Y, Fukuhara S, Morino S, et al. Structural features of human initiation factor 4E, studied by X-ray crystal analyses and molecular dynamics simulations. J Mol Biol. 2003;328(2):365–83.View ArticlePubMedGoogle Scholar
- Mader S, Lee H, Pause A, Sonenberg N. The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Mol Cell Biol. 1995;15(9):4990–7.PubMed CentralPubMedGoogle Scholar
- Altmann M, Schmitz N, Berset C, Trachsel H. A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E. Embo J. 1997;16(5):1114–21.View ArticlePubMed CentralPubMedGoogle Scholar
- Li L, Hong R, Hastings J. Three functional luciferase domains in a single polypeptide chain. Proc Natl Acad Sci. 1997;94:8954–8.View ArticlePubMed CentralPubMedGoogle Scholar
- Lasko P. Posttranscriptional regulation in Drosophila oocytes and early embryos. Wiley Interdiscip Rev RNA. 2011;2(3):408–16.View ArticlePubMedGoogle Scholar
- Lasda EL, Blumenthal T. Trans-splicing. Wiley Interdiscip Rev RNA. 2011;2(3):417–34.View ArticlePubMedGoogle Scholar
- Jankowska-Anyszka M, Lamphear BJ, Aamodt EJ, Harrington T, Darzynkiewicz E, Stolarski R, et al. Multiple isoforms of eukaryotic protein synthesis initiation factor 4E in Caenorhabditis elegans can distinguish between mono- and trimethylated mRNA cap structures. J Biol Chem. 1998;273(17):10538–42.View ArticlePubMedGoogle Scholar
- Keiper BD, Lamphear BJ, Deshpande AM, Jankowska-Anyszka M, Aamodt EJ, Blumenthal T, et al. Functional characterization of five eIF4E isoforms in Caenorhabditis elegans. J Biol Chem. 2000;275(14):10590–6.View ArticlePubMedGoogle Scholar
- Dinkova TD, Keiper BD, Korneeva NL, Aamodt EJ, Rhoads RE. Translation of a small subset of Caenorhabditis elegans mRNAs is dependent on a specific eukaryotic translation initiation factor 4E isoform. Mol Cell Biol. 2005;25(1):100–13.View ArticlePubMed CentralPubMedGoogle Scholar
- Zhang H, Campbell DA, Sturm NR, Lin S. Dinoflagellate spliced leader RNA genes display a variety of sequences and genomic arrangements. Mol Biol Evol. 2009;26(8):1757–71.View ArticlePubMed CentralPubMedGoogle Scholar
- Hearne JL, Pitula JS. Identification of two spliced leader RNA transcripts from Perkinsus marinus. J Eukaryot Microbiol. 2011;58(3):266–8.View ArticlePubMedGoogle Scholar
- Zhang S, Sui Z, Chang L, Kang K, Ma J, Kong F, et al. Transcriptome de novo assembly sequencing and analysis of the toxic dinoflagellate Alexandrium catenella using the Illumina platform. Gene. 2014;537(2):285–93.View ArticlePubMedGoogle Scholar
- Hernandez G, Vazquez-Pianzola P. Functional diversity of the eukaryotic translation initiation factors belonging to eIF4 families. Mech Dev. 2005;122(7–8):865–76.View ArticlePubMedGoogle Scholar
- Freire ER, Malvezzi AM, Vashisht AA, Zuberek J, Saada EA, Langousis G, et al. Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog. Eukaryot Cell. 2014;13(7):896–908.View ArticlePubMed CentralPubMedGoogle Scholar
- Freire ER, Vashisht AA, Malvezzi AM, Zuberek J, Langousis G, Saada EA, et al. eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei. RNA. 2014;20(8):1272–86.View ArticlePubMedGoogle Scholar
- Volpon L, Osborne MJ, Culjkovic-Kraljacic B, Borden KL. eIF4E3, a new actor in mRNA metabolism and tumor suppression. Cell Cycle. 2013;12(8):1159–60.View ArticlePubMed CentralPubMedGoogle Scholar
- Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29(7):644–52.View ArticlePubMed CentralPubMedGoogle Scholar
- Park MG, Cooney SK, Yih W, Coats DW. Effects of two strains of the parasitic dinoflagellate Amoebophrya on growth, photosynthesis, light absorption, and quantum yield of bloom-forming dinoflagellates. Mar Ecol Prog Ser. 2002;227:281–92.View ArticleGoogle Scholar
- Bachvaroff TR, Place AR, Coats DW. Expressed Sequence Tags from Amoebophrya sp. Infecting Karlodinium veneficum: Comparing Host and Parasite Sequences. J Eukar Microbiol. 2009;56(6):531–41.View ArticleGoogle Scholar
- Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011;7:539.View ArticlePubMed CentralPubMedGoogle Scholar
- Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25.View ArticlePubMed CentralPubMedGoogle Scholar
- Thorvaldsdottir H, Robinson JT, Mesirov JP. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 2013;14(2):178–92.View ArticlePubMed CentralPubMedGoogle Scholar
- Stamatakis A. RAxML-VI-HPC: Maximum likelihood-based phylogenetic analysis with thousands of taxa and mixed models. Bioinformatics. 2006;22(21):2688–90.View ArticlePubMedGoogle Scholar
- Schmidt HA, Strimmer K, Vingron M, von Haeseler A. TREE-PUZZLE: maximum liklihood phylogenetic analysis using quartets and parallel computing. Bioinformatics. 2002;18:502–4.View ArticlePubMedGoogle Scholar
- Vacic V, Uversky VN, Dunker AK, Lonardi S. Composition Profiler: a tool for discovery and visualization of amino acid composition differences. BMC Bioinformatics. 2007;8:211.View ArticlePubMed CentralPubMedGoogle Scholar
- Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: A sequence logo generator. Genome Res. 2004;14:1188–90.View ArticlePubMed CentralPubMedGoogle Scholar
- Kelley LA, Sternberg MJ. Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc. 2009;4(3):363–71.View ArticlePubMedGoogle Scholar
- Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14(1):33–8. 27–38.View ArticlePubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.