In and out of the rRNA genes: characterization of Pokey elements in the sequenced Daphnia genome
© Elliott et al.; licensee BioMed Central Ltd. 2013
Received: 18 July 2013
Accepted: 29 August 2013
Published: 23 September 2013
Only a few transposable elements are known to exhibit site-specific insertion patterns, including the well-studied R-element retrotransposons that insert into specific sites within the multigene rDNA. The only known rDNA-specific DNA transposon, Pokey (superfamily: piggyBac) is found in the freshwater microcrustacean, Daphnia pulex. Here, we present a genome-wide analysis of Pokey based on the recently completed whole genome sequencing project for D. pulex.
Phylogenetic analysis of Pokey elements recovered from the genome sequence revealed the presence of four lineages corresponding to two divergent autonomous families and two related lineages of non-autonomous miniature inverted repeat transposable elements (MITEs). The MITEs are also found at the same 28S rRNA gene insertion site as the Pokey elements, and appear to have arisen as deletion derivatives of autonomous elements. Several copies of the full-length Pokey elements may be capable of producing an active transposase. Surprisingly, both families of Pokey possess a series of 200 bp repeats upstream of the transposase that is derived from the rDNA intergenic spacer (IGS). The IGS sequences within the Pokey elements appear to be evolving in concert with the rDNA units. Finally, analysis of the insertion sites of Pokey elements outside of rDNA showed a target preference for sites similar to the specific sequence that is targeted within rDNA.
Based on the target site preference of Pokey elements and the concerted evolution of a segment of the element with the rDNA unit, we propose an evolutionary path by which the ancestors of Pokey elements have invaded the rDNA niche. We discuss how specificity for the rDNA unit may have evolved and how this specificity has played a role in the long-term survival of these elements in the subgenus Daphnia.
KeywordsTransposons Daphnia Pokey Ribosomal DNA Insertion specificity
Transposable elements (TEs) are found in nearly all organisms and often comprise substantial portions of eukaryotic genomes . Many TEs insert into locations throughout the genome, while others insert preferentially into specific sequences. A site preferred by non-long terminal repeat (non-LTR) retrotransposons is the locus encoding rRNA . Pokey is the only example of a DNA transposon known to insert specifically in rDNA. Pokey inserts into the same 28S gene region that is highly targeted by non-LTR elements . Insertion of any of these elements is expected to disrupt the production of functional rRNA from the inserted units.
rDNA is comprised of hundreds to thousands of units arrayed in tandem encoding one copy each of the core 18S, 5.8S and 28S rRNAs. The many copies of each rRNA gene show high sequence identity, the product of recombinational processes termed concerted evolution (reviewed in ). The primary mechanism conferring high identity between copies is unequal crossing over, which also generates the large variation in rDNA copy number observed between members of the same species . The combined processes of concerted evolution and selection against inserted units require that any element with a long-term presence in the rDNA unit regularly generate new insertions to avoid being eliminated from the locus [4, 5].
Pokey elements are members of the piggyBac superfamily of DNA-mediated TEs that insert into TTAA target sequences [3, 6]. This element was first identified in the cladoceran crustacean Daphnia pulex, and is now known to be widespread throughout the subgenus Daphnia. Unlike most DNA TEs, Pokey elements have undergone stable vertical inheritance for millions of years . To the best of our knowledge, the only other organisms in which open reading frames (ORFs) similar to those in Pokey have been found are the silkmoth Bombyx mori, the tunicate Ciona savignyi, and the rotifer Adineta vaga. Pokey elements have been found at multiple TTAA insertion sites throughout the genome and thus, like other piggyBac elements, appear to require little additional conservation of target sites [3, 11]. Nevertheless, Pokey have been repeatedly found at just one location in the 28S genes despite the presence of over 30 TTAA motifs in the entire rDNA unit. While this finding might suggest that properties in addition to TTAA are preferred for Pokey insertion, the frequency of independent Pokey insertions in the rDNA locus is not known. Thus, it is unclear whether rDNA acts as a sink or source for Pokey elements, or whether there is free and on-going exchange between Pokey elements in and outside the rRNA genes.
In this study, we used the original sequencing reads available from the Daphnia genome sequencing project, available at the Trace Archives at GenBank, as well as the annotated scaffold sequences to study Pokey elements and their interactions with 28S genes. The Pokey elements are divided into two divergent lineages each possessing a unique inverted terminal repeat (ITR) structure. Both lineages carry repeated copies of a segment from the intergenic spacer (IGS) region of the rDNA unit. In addition, two lineages of non-autonomous miniature inverted repeat transposable elements (MITEs) are present at the Pokey site in 28S genes, and elsewhere in the genome. Finally, weak target sequence preferences for Pokey and the MITEs were found that are consistent with the site that is targeted in the 28S gene. We suggest that Pokey elements have evolved specificity for their 28S gene insertion site and their presence at this site has played a key role in their long term survival in Daphnia by acting as a source for Pokey and their MITEs throughout the genome.
rDNA sequence variation
Concerted evolution is expected to maintain very high sequence identity among all copies of the rDNA unit. The rDNA transcription units (external transcribed spacer through the 28S gene) in sequenced genomes of Drosophila and Nasonia contain from 3 to 18 sites in which sequence variants are present in over 3% of units. In contrast, the Daphnia rDNA transcription unit and a 500 bp non-repetitive region from the IGS contain no sequence variants at the 3% threshold. This especially low level of rDNA variation is consistent with the very high level of homozygosity at allozyme and microsatellite markers observed in the sequenced Daphnia isolate , the low level of sequence variation in 28S genes from D. pulex in natural populations , and the high rate of recombination observed in the rDNA of a closely related species, Daphnia obtusa.
Pokey elements in 28S genes and the genome
A consensus sequence for Pokey copies was assembled from the original sequence reads of the D. pulex genome (Additional file 1). We also identified 69 elements containing intact ITRs at both ends from the annotated genome scaffolds at wFleabase. We aligned these sequences to two copies of Pokey elements from D. pulicaria rDNA, which were designated pcPokey S (5 kb) and pcPokey L (6.6 kb) . As shown in Figure 1B, the Pokey elements contain either 12 or 16 bp imperfect ITRs, a 5′ non-coding region (NCR), an ORF encoding a putative transposase, and a 3′ NCR. The D. pulex copies were up to 9,800 bp in length (Additional file 2) with the majority of the length variation occurring in the 5′ NCR (discussed below). Excluding this repetitive region, the canonical Pokey element is approximately 4,500 bp.
We also identified an additional 91 incomplete sequences from 400 to 4,400 bp in length that lack either the 5′ or 3′ ITR, or both. The total number of Pokey elements based on these genomic searches was 160, similar to the 175 estimated by comparing the depth of coverage of Pokey sequence reads to the average coverage of single copy genes . We estimate that six of the 175 copies are inserted into 28S genes and they all have the 12-bp ITRs.
Like the Pokey elements, MITEs found outside 28S genes also target TTAA sites, suggesting that they use the transposase of Pokey elements. We hereafter refer to these MITEs as mPok. About 25 to 30 copies of these mPok were found in 28S genes, all with 12-bp ITRs. The total genome contains 90 to 110 copies with 60 mPok sequences in the assembled scaffolds (Additional file 2).
Cluster analysis of Pokey and mPok elements
Sequence divergence between Pokey elements from the Daphnia genome
m Pok 1
m Pok 2a
m Pok 2b
Characterization of the Pokey transposase
The ORF from the pcPokey L element was originally reported by Penton and colleagues  to be 1,461 bp encoding a protein of 487 amino acids. However, this coding region was suggested to contain a 68 bp intron (Y Bigot, personal communication), which when spliced from an RNA transcript would enable the production of a 668 amino acid protein. Pokey elements from D. pulex also appear to have this intron, which ranged in size from 68 to 74 bp in Pokey A and from 79 to 84 bp in Pokey B. Analysis of Pokey RNA transcripts by RT-PCR confirmed that the putative intron sequence can be spliced out .
Pokey A and Pokey B transposase genes encode conserved motifs shared among the transposase genes of diverse piggyBac elements . These include a DDD (aspartic acid) motif (amino acid residues 436, 544 and 659) that is considered essential for transposase activity, an imperfect zinc finger motif that is believed to be either a chromatin-interacting Plant Homeo Domain or a protein-protein interaction domain, and a putative nuclear localization signal. Keith and colleagues  identified a fourth D residue C-terminal to the catalytic DDD triad. When they mutated this charged D to an uncharged N (asparagine) in a piggyBac construct, they observed a significant reduction in the transposition rate. This fourth residue is N instead of D in the D. pulicaria and D. pulex Pokey elements (Additional file 4). Partial sequences of Pokey transposase genes from other species in the subgenus Daphnia all encode an N at this site.
Of the 69 elements identified from the assembled scaffolds, two Pokey A and two Pokey B elements were identified that may encode transposition-competent transposases (identified on the NJ tree in Figure 3). The ORF of these elements lacked premature stop codons and contained all features known or inferred to be important for the transposition of piggyBac.
Repeated sequences in Pokey and mPok
In addition to A and C repeats, there are three other short repeated sequences in the 5′ and 3′ NCR of Pokey elements from the Daphnia genome that are shared with the mPok elements (Figure 2 and Additional file 6). Some of these repeats may correspond to repeat sequences previously found in other piggyBac elements, such as the one diagrammed in Additional file 6 from Trichoplusia ni.
Target site preferences for Pokey and mPok
Target site duplications flanking Pokey elements in the Daphnia genome sequence
Target site duplications
% of insertions
Pokey diversity in the Daphnia genome
Analysis of over 160 Pokey and Pokey-like sequences from the D. pulex genome revealed four well-supported clusters. Two clusters of larger elements with an average size of 5,100 bp were designated Pokey A and Pokey B. The clusters have diverged in sequence by about 40%, have different ITR structures and include members that possess an intact transposase ORF. The two other clusters are MITEs, designated mPok 1 and mPok 2, because each mPok element contains an ITR and other non-coding sequences corresponding to one of the full-length Pokey elements. Annotated Pokey B and mPok 2 elements outnumber Pokey A and mPok 1 elements by over 4:1.
Available evidence suggests that both Pokey A and Pokey B occur in D. obtusa and thus the two lineages have likely persisted across multiple speciation events. Vertical diversification of TEs within the same genome can be driven by drift, selection, or more likely a combination of the two. Two models have been proposed. Lampe and colleagues  observed a loss of interaction between the ITRs and transposases of Tc1/mariner elements from different subfamilies with sequence divergence greater than 16%. They postulated that silencing mechanisms based on sequence similarity might create intragenomic selection that favors divergence of the transposase and ITR sequences of related TEs to escape silencing. A second possibility is that the presence of numerous non-autonomous elements drives the divergence of transposase and ITR sequences because the non-autonomous copies titrate the transposase from autonomous copies and decrease their fitness . In that case, intragenomic selection might favor divergent elements whose transposases can only recognize their own ITRs.
The ability of Pokey A and Pokey B elements to cross-mobilize could be investigated using yeast excision, yeast one-hybrid and/or electrophoretic mobility shift assays to determine the strength of interaction between the transposases and ITRs of each group. Although the differences in sequence between the two ITR structures appear minor (Figure 1), Casteret and colleagues  demonstrated that a small number of single nucleotide changes to the ITR of the drosophilid DNA transposon Mos1 produced significant changes in transposition rate.
The mPok elements appear to be of an atypically large size (approximately 750 bp) compared to other MITEs, which can be as small as around 130 bp . However, MITEs that are even larger than mPok have now been discovered in phylogenetically diverse eukaryotes (reviewed in ) suggesting that large MITEs are more common than once thought. One mechanism to explain the origin of large MITEs is progressive internal deletion of autonomous DNA TEs and subsequent selection for increasing transposition rate among the resultant elements over time . Thus, the larger size of mPok elements could be a consequence of their recent evolution. While this could be true for the mPok1 elements, which show little sequence diversity, the occurrence of highly divergent mPok 2b copies is not consistent with a recent origin (Figure 4). Indeed, Deprá and colleagues  suggested that the Mar MITEs in Drosophila willistoni, which are similar in size to the mPok elements, may have originated prior to the diversification of the willistoni subgroup 5.7 MYA, suggesting that large size does not necessarily indicate recent origin.
Repeated sequences in Pokey
An unusual aspect of the Pokey A and B lineages in Daphnia is the presence of sequences derived from NCRs of the rDNA unit. This includes an approximately 200 bp sequence from a non-repetitive region of the IGS (A repeats) and an approximately 50 bp sequence from ITS2 (C repeats) (Figures 1 and 5). Pokey elements contain from 2 to 5 copies of these rDNA sequences within their 5′ NCR (Figure 5). The highly recombinogenic nature of these repeats within the Pokey elements was first suggested by their differential spacing in pcPokey S and pcPokey L  and is strongly supported by this analysis in which particular combinations of A and C repeats are unique to only one or a few Pokey elements.
The acquisition of DNA to the 5′ NCR of Pokey does not appear to be limited to rDNA. For example, Pokey 62 contains a unique, approximately 3,600 bp sequence of which approximately 1,100 bp is derived from sequence on a non-rDNA scaffold in the Daphnia genome. Thus, Pokey elements often acquire sequences from their host’s genome. Langer and colleagues  proposed that Ds elements could acquire host sequence if the transposase slides after binding but before cutting, or if cryptic ITR-like sequences exist downstream of an element. However, the acquisition of sequences well within the 5′ NCR of the Pokey elements argues against such a simple explanation (Figure 5).
What is the significance of the A and C repeats? It is possible that they have no function and that their origin was chance recombination events that had no fitness impact on Pokey. However, the finding that all but one copy of Pokey from both lineages contain these repeats suggests that they do play some role in Pokey activity. Possible functions of these sequences include transcription enhancers, transcription terminators to prevent the formation of aberrant rRNA read-through transcripts, or binding sequences to recruit epigenetic modifiers [34–36]. We suggest a transcription role for these repeats to be most likely as mPok elements, which do not need to be transcribed to be mobilized by a Pokey transposase, do not have the rDNA repeats.
The most remarkable property of the A repeats is that the same sequence was retained in both the Pokey A and B lineages. Not only do the A repeats correspond to the highest level of sequence conservation between the two lineages, but the A repeats within the two Pokey lineages are as well conserved as IGS sequences undergoing concerted evolution within the rDNA unit (Figure 6). This high level of sequence identity suggests that recombination between the Pokey repeats and the rDNA repeats occurs on a regular basis, thus strengthening the argument that Pokey elements have become highly specialized for their insertion into the rDNA locus.
Target site selection and the rDNA niche
While it is not possible to assemble the sequences of individual Pokey elements inserted in rDNA, it should be noted that the consensus Pokey sequence from rDNA is similar to an assembled non-rDNA copy that could putatively encode a functional transposase (Figure 3). Given the rapid turnover of rDNA units, Pokey elements within the locus should be among the newest insertions, while those outside of rDNA are a combination of new and old insertions.
Pokey is the only DNA-mediated TE that is known to evolve insertion specificity for the rDNA unit. Remarkably, the Pokey insertion site is in the same region of the 28S gene that is also the target site for a number of non-LTR retrotransposons (reviewed in ). Two of these elements, R2 and R5, which insert within a few base pairs of the Pokey site, encode related endonucleases that have an active site similar to class IIS restriction enzymes [37, 38]. The R2 endonuclease has been shown to have exceptional specificity for the 30 to 40 nucleotides surrounding its insertion site [39, 40]. Two other non-LTR retrotransposons, R1 and R4, insert 75 and 28 bp, respectively, downstream of the Pokey site. These elements encode an endonuclease with similarity to the apurinic endonuclease involved in DNA repair . The endonuclease encoded by R1 has also been shown to have sequence specificity for the insertion site [41, 42]. In all four cases, most copies of the element are inserted in rDNA with most copies outside rDNA inserted into sites with sequence similarity to the 28S gene target site [13, 43].
The transposase of Pokey elements represents a third protein that has evolved specificity for this region of the 28S gene. Some of the best-studied examples of integrases that have evolved insertion specificity involve the LTR retrotransposons of yeast [44–47]. In these cases, the integrases have evolved protein-protein specificity for association with specific transcription factors or chromatin structural components rather than actual DNA sequence specificity. Such protein-chromatin interactions could also be involved in the insertion specificity of Pokey elements, but we are not aware of any specific chromatin components that are bound to the central region of 28S genes. Alternatively, the A repeat associated with Pokey elements may contain a recognition site for a nucleolar protein that helps guide into the nucleolus Pokey elements that have been excised and are ready for insertion.
It seems a remarkable coincidence that three different lineages of TEs have evolved specificity for the same small region of the rDNA unit. The 28S target region is highly conserved, but there are many regions of the 18S and 28S genes that are conserved across eukaryotes. We suggest either the DNA in this region is highly exposed and thus accessible to the TE machinery, a yet unknown chromatin component can be utilized by the TE in its evolution of specificity, or this is one of only a few areas of the rDNA where a TE can insert without being quickly eliminated by recombination or selected against by the synthesis of disrupted rRNA.
Based on the concordance between phylogenies of rDNA Pokey elements and their hosts, Penton and Crease  concluded that Pokey has undergone stable, vertical inheritance in the rDNA of species in the subgenus Daphnia since its origin. Thus, unlike most Class II TEs, Pokey elements appear to have evaded complete silencing by the host for millions of years. The unique breeding system of Daphnia, involving extended periods of apomictic reproduction, and the complete loss of sexuality in some lineages may have created strong selection pressure on ancestral Pokey elements to avoid causing deleterious mutations in their host, while still maintaining a transposition rate high enough to survive. The theory describing the interaction between TEs and asexual or partially asexual hosts predicts three possible outcomes: (1) active elements are lost, (2) the host goes extinct due to TE-induced mutation, or (3) the elements become domesticated and the threat is neutralized . However, Pokey’s invasion of rDNA suggests a fourth outcome, the long-term persistence of active elements.
Zhou and colleagues  have argued that rDNA is an ideal TE niche, because it is difficult for the host to completely silence elements that have inserted into genes that must be expressed. In addition, TEs inserted in the locus are continually removed by recombination events so old copies that could interfere with the elements are eliminated. Finally, each insertion has a predictable, small effect on the fitness of the host. This effect is small because all organisms contain more than enough rDNA for the production of rRNA, and those rDNA units with insertions are usually not transcribed . R2 and R1 elements, which are abundant in the rDNA of arthropods including crustaceans, have not been found in Daphnia. Perhaps Pokey elements are even better adapted for this niche in that they can be lost from the rDNA locus, but copies located outside the rDNA can on occasion be active and re-establish insertions in the locus. Indeed, individual D. pulex that lack Pokey A in rDNA have been observed, but no individuals have been observed that completely lack Pokey elements [11, 19, 50–52].
In spite of what would appear to be a seemingly inhospitable location for a DNA transposon, Pokey has evolved specificity for a site in the 28S genes of Daphnia. Analysis of both the annotated D. pulex genome and the raw trace files revealed that rDNA units display extremely low levels of sequence variation consistent with the high rates of recombination previously observed for this locus. Indeed, Pokey has diversified into two lineages of autonomous elements, Pokey A and Pokey B, which appear to have persisted across multiple speciation events. While members of the B lineage are located in the rDNA of the population in Oregon that was selected for genomic sequencing , members of the A lineage are in the rDNA of D. pulicaria and D. pulex populations outside Oregon [3, 7, 52]. Both Pokey lineages have given rise to two parallel lineages of MITES, mPok 1 and mPok 2, which appear to be deletion derivatives of the full-length elements.
Part of the specificity of Pokey elements can be attributed to the sequence specify of the transposase itself, as the target site of non-rDNA copies bears weak sequence similarity to the 28S rRNA insertion site. However, both Pokey lineages possess repeat sequences derived from rDNA that vary in arrangement and copy number. These repeats may play a role in the expression of Pokey elements from the rDNA locus, and/or a role in insertion specificity. Whatever their function, the Pokey repeats are evolving in concert with each other and with the rDNA unit itself suggesting ongoing sequence exchange. It remains unknown whether Pokey elements in or out of the rDNA locus are most active, and what fraction of new insertions occur in rDNA. While more insertions are found outside rDNA, this could simply reflect the fact that non-rDNA insertions are more stable over time. Overall, our results suggest a complex interaction between Pokey and its host, and highlight the need to concentrate not only on host traits but also on traits of individual families when trying to understand the current dynamics and past evolutionary history of TEs.
Search for and assembly of rDNA and Pokey elements
The original sequencing reads of the genome sequencing project from the cladoceran crustacean Daphnia pulex were accessed by basic local alignment search tool (BLAST) [53, 54] in the Trace Archives at GenBank . In addition, BLAST searches were conducted of the assembled scaffolds at wFleaBase .
The search for Pokey elements in 28S genes was conducted in the same manner as searches for other 28S-specific TEs in rDNA . Briefly, a BLAST search was conducted using the downstream region flanking the Pokey insertion site as the query. Reads identified in this search were examined upstream of the query region for sequences that were not 28S and thus putative TEs. Once the consensus of the TE end was acquired, iterative BLAST searches were conducted using the end of each newly acquired TE extension until the 5′ junction of the element with the 28S gene was reached. In order to identify copies present outside 28S genes, the ends of the TE consensus sequences were used as BLAST queries and the flanking sequences examined. Sequences of the putative transposase gene were analyzed using the PSORTII server  to identify features of the amino acid sequence.
Cluster analysis of Pokey elements
Pokey elements were aligned using a combination of the CLUSTAL, MUSCLE and MAFFT multiple sequence alignment programs available from the EMBL-EBI website . Alignments were manually adjusted in the program BioEdit . Only sequences with less than 5% ambiguous bases across the aligned region and containing an ITR at both ends were used in cluster analyses. Measurements of pairwise sequence divergence were calculated using the Kimura 2-parameter method  in MEGA4 . NJ trees  were also constructed in MEGA4. Bootstrap analysis was performed on 1000 pseudo-replicates for each tree . The alignment of full-length elements excluded the variable repeat region between the 5′ ITR and the transposase gene. In addition, a dataset including the last approximately 1,600 bp of the 3′ end of rDNA Pokey elements from species in the subgenus Daphnia was aligned with the Pokey elements from the Daphnia genome sequence and used to generate an NJ tree.
Sequence variation in rDNA and Pokey elements
Sequence variation present in the rDNA transcription units and in a 500 bp region of the IGS was evaluated in the same manner as described by Stage and Eickbush . Briefly, 525 bp overlapping regions of each consensus were used as BLAST queries in the trace archives. Approximately 250 reads were collected from each BLAST search and evaluated for sequence changes present in at least eight sequence reads. In order to screen out sequencing errors, sites containing sequence differences were further evaluated using the trace quality scores available through the trace archives at GenBank .
A total of 26 base pairs on each side of the Pokey insertion site of both Pokey and mPok elements, all oriented in the 5′ to 3′ direction, were compared to determine if a preferred base is present at each position. A graphical representation of sequence conservation was made using WebLogo . Only the 4 bp upstream and 15 bp downstream of the insertion contain preferred bases.
Analysis of repeat sequences in Pokey
Identification of repeat sequences within Pokey, and comparisons between Pokey and rDNA were performed using Pustell DNA matrix in MacVector 10.0 (MacVector Inc., Cary, NC, USA). Default parameters were used with 80% sequence identity in a 16 bp window.
Basic local alignment search tool
Inverted terminal repeat
Long terminal repeat
Miniature inverted repeat transposable element
Pokey-derived miniature inverted repeat transposable element
Open reading frame
Reverse transcriptase polymerase chain reaction
Funding for this work was provided by a grant from the NIH (GM42790) to THE and an NSERC Discovery Grant to TJC.
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