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Conceived and designed the experiments: SW LZ. Performed the experiments: SW. Analyzed the data: SW LZ EM MVM. Wrote the paper: SW LZ EM MVM.
Miniature inverted-repeat transposable elements (MITEs), which are common in eukaryotic genomes, are small non-coding elements that transpose by utilizing transposases encoded by autonomous transposons. Recent genome-wide analyses and cross-mobilization assays have greatly improved our knowledge on MITE proliferation, however, specific mechanisms for the origin and evolution of MITEs are still unclear.
A group of coral MITEs called CMITE were identified from two corals, Acropora millepora and Acropora palmata. CMITEs conform to many common characteristics of MITEs, but also present several unusual features. The most unusual feature of CMITEs is conservation of the internal region, which is more conserved between MITE families than the TIRs. The origin of this internal region remains unknown, although we found one CMITE family that seems to be derived from a piggyBac-like transposon in A. millepora. CMITEs can form tandem arrays, suggesting an unconventional way for MITEs to increase copy numbers. We also describe a case in which a novel transposable element was created by a CMITE insertion event.
To our knowledge, this is the first report of identification of MITEs from coral genomes. Proliferation of CMITEs seems to be related to the transposition machinery of piggyBac-like autonomous transposons. The highly conserved internal region of CMITEs suggests a potential role for this region in their successful transposition. However, the origin of these unusual features in CMITEs remains unclear, and thus represents an intriguing topic for future investigations.
Transposable elements (TEs) are prevalent in the genomes of all animals and plants, and are often thought of as selfish or parasitic elements . The relationship between TEs and their hosts has been described as an arms race, with the TEs trying to increase their copy number in the host genome and the host trying to protect the integrity of its genetic content . This arms race can lead to enhanced genome plasticity and thus drive host genome evolution (for recent reviews, see , ).
Eukaryotic TEs can be divided into two major classes, retrotransposons (class I) and DNA transposons (class II), on the basis of the presence or absence of RNA as a transposition intermediate . With few exceptions, classic “cut-and-paste” DNA transposons have terminal inverted repeats (TIRs) at both ends and transpose using the so-called “cut-and-paste” mechanism (for a review, see ). Some DNA transposons are autonomous, encoding their own transposases, while others are nonautonomous. Nonautonomous DNA transposons maintain transposition activity by retaining the cis sequences (e.g. TIRs or in some cases, subterminal repeated sequences) recognized by trans transposases from autonomous DNA transposons.
Miniature inverted-repeat transposable elements (MITEs) are a special class of nonautonomous DNA transposons that can transpose by “borrowing” the transposition machinery of autonomous DNA transposons with similar TIR signals –. MITEs have a suite of well known characteristics such as small size (usually less than 500 bp), conserved TIRs, and the absence of protein-coding sequences . In contrast to typical nonautonomous DNA transposons, MITEs are highly homogeneous in size and are usually present in genomes in very high copy numbers. Because MITEs do not encode transposases, their classification is mainly based on shared TIR and target site duplication (TSD) sequences. To date, most MITEs can be classified into seven superfamilies that include Tc1/mariner (Stowaway-like MITEs), PIF/Harbinger (Tourist-like MITEs), piggyBac/TTAA and hAT . Although recent genome-wide analyses and cross-mobilization assays have greatly improved our knowledge on MITE proliferation , , –, specific mechanisms for the origin and evolution of MITEs are still unclear.
Here, we present the first report of a group of coral MITEs called CMITE, which were identified from whole-genome shotgun (WGS) sequences of two coral species, Acropora millepora and Acropora palmata. Although CMITEs conform to many common characteristics of MITEs, they also present the following unusual features: (i) highly conserved internal region but less conserved TIRs, (ii) formation of tandem arrays, and (iii) de novo assembly of a novel TE.
WGS sequences of A. millepora and A. palmata were downloaded from the National Center for Biotechnology Information (NCBI) database. There were 14625 and 11024 entries for A. millepora and A. palmata, respectively.
CMITEs with matching TIRs (13~14 bp in length) were first identified using the FINDMITE program . In order to search for possible related elements, a 60 bp consensus sequence (5′-AGGGGTTCCCCATTGACGAGTAAAATCGTCTGGCGTTAGACAGAGTAAAATCTATAAGTG-3′) from the internal conserved region of CMITEs was used for blastn search . A cutoff value of e ≤10−5 was used as the significance threshold for the comparison.
Multiple sequence alignment was performed using the MegAlign program (part of the DNASTAR software package) and sequence alignments were manually refined. A formula was adopted to estimate the copy number of CMITEs in the genome: copy number = (number in database × genome size)/database size . This calculation was only possible for A. millepora, since there is a previously published estimate of 200 Mbp for this genome size .
Using piggyBac-like elements from RepBase 13.05 (n=73, ) as queries (tblastx, e≤10−4), we identified 14 distinct A. millepora piggyBac-like sequences. Eleven of them came from the A. millepora larval transcriptome (NCBI ID: SRA003728, ), and 3 came from the WGS sequences.
Two approaches were used to isolate piggyBac-like transposons from A. millepora genome. In the first approach (direct PCR), polymerase chain reaction (PCR) primers were designed based on the TIR sequences of CMITE family I, II and III in an effort to isolate MITE family-specific piggyBac-like transposons. PCR amplifications were set up in a 20 µL volume composed of 10 ng A. millepora genomic DNA, 0.5 µM each primer, 0.2 mM dNTP, 1× Phusion HF buffer and 0.4 U Phusion hot start high-fidelity DNA polymerase (NEB, Ipswich, MA) in a DNA Engine Tetrad 2 thermal cycler (Bio-Rad, Hercules, CA). All cycling began with an initial denaturation at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 60°C for 30 s, 72°C for 5 min, and a final extension at 72°C for 10 min. PCR products were detected by agarose gel electrophoresis. PCR product containing fragments in the desired size range (i.e. 2–6 kb) was purified using QIAquick PCR purification kit (Qiagen, Valencia, CA). Because Phusion DNA polymerase generates blunt-end PCR products, 3′ A overhangs must be added to the blunt PCR product before TA cloning. The A-addition reaction was set up in a 10 µL volume composed of ~200 ng purified PCR product, 0.2 mM dATP, 1× ThermoPol buffer and 1 U Taq DNA polymerase (NEB, Ipswich, MA), and incubated at 72°C for 30 min. After treatment, PCR products were ligated into pGEM-T vector (Promega, Madison, WI) and subsequently transformed into TOP10 competent Escherichia coli cells (Invitrogen, Carlsbad, CA). Recombinant clones were screened for inserts of correct size, and then were sequenced at the DNA Core Facility at UT Austin. In this approach, the exact TIR sequences of a piggyBac-like element remain unknown since the TIR region of this element serves as a primer-binding site. An adaptor-ligation PCR method  was utilized to obtain the TIR sequences of a given piggyBac-like element. To prepare the adaptor-ligated DNA, 200 ng of A. millepora genomic DNA was digested with 5 U MseI (NEB, Ipswich, MA) at 37°C for 3 h. The reaction was inactivated at 65°C for 20 min. A ligation solution containing 50 pMol MseI-adapter (5′ CAGCAGACTTGAGGTCGTGGTGCTGAGTGCAGTG 3′ and 5′ TACACTGCACTCAGC-NH2 3′), 200 U T4 DNA ligase (NEB, Ipswich, MA) and 1 mM ATP (NEB, Ipswich, MA) was added, and the resultant solution was incubated at 16°C for 16 h. PCR amplifications were set up in a 20 µL volume composed of 10 ng adaptor-ligated DNA, 0.1 µM adaptor-specific primer (5′ GCCTTGCCAGCCCGCTTGTCAGCAGACTTGAGGTCGTGGT 3′), 0.1 µM transposon-specific upstream or downstream primer, 0.2 mM dNTP, 1× Advantage 2 PCR buffer and 1× Advantage 2 Polymerase Mix (Clontech, Mountain View, CA). All cycling began with an initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, 68°C for 30 s, and a final extension at 68°C for 10 min. PCR products were then cloned and sequenced as described above.
In the second approach, inverse PCR was utilized in an effort to isolate full piggyBac-like transposons based on the 14 A. millepora piggyBac-like sequences. A 600-ng aliquot of A. millepora genomic DNA was digested with 5 U NcoI, BglII and BamHI (NEB, Ipswich, MA) respectively at 37°C for 3 h. Digested DNA was purified using QIAquick PCR purification kit (Qiagen, Valencia, CA), and was self-circularized in a final volume of 300 µL using T4 DNA liagse (NEB, Ipswich, MA) at 16°C for 16 h. After purification, ~10 ng of ligated DNA was used for PCR amplification. PCR amplification, TA cloning and sequencing were followed the same procedure in the direct PCR approach. Primers used in the two approaches were designed based on several principles as described by Matz  so that all PCR amplifications could be achieved at the same annealing temperature.
Transposase protein sequences were aligned using the ClustalW method . The protein sequence alignment is available in the Supplementary Dataset S1. Phylogenetic analysis was performed with the program MrBayes 3.1 . The appropriate model of evolution was identified as WAG+G+I  using the MCMC model-jumping method. The MCMC chain was run for 1,000,000 generations with a sample frequency of 200. In total, 5000 trees were produced, of which the first 4500 were discarded as burn-in while summarizing the data.
When searching for MITEs in the WGS sequences of A. millepora, our attention was quickly turned to several predicted MITEs (which we later called CMITE), which had different TIRs but shared highly conserved sequences in their internal region. Using the FINDMITE program , eight CMITE elements with matching TIRs (13~14 bp in length) were initially identified in the WGS sequences of A. millepora and A. palmata. These CMITEs showed many of the characteristic features of MITEs. They were small (about 100 bp) and homogeneous in size. They had TIRs and were flanked by TTAA TSDs. In contrast to most other MITEs, however, the 75-base-long internal region of CMITEs was remarkably well conserved across CMITE families (Fig. 1). Based on the similarity of their TIRs, eight CMITE elements can be classified into three families (family I, II and III) (Table 1), which is also correlated with the variations in their internal regions, except for one case: AP824033492 had a family II-like internal region.
To identify possible related elements, we used a 60 bp consensus sequence from the most invariant part of the internal region as a query in blastn search against the WGS sequences of A. millepora and A. palmata. This search identified 88 significant hits from 78 different A. millepora WGS entries, and 111 from 94 different A. palmata WGS entries. Multiple matches were found in 7 and 12 WGS entries of A. millepora and A. palmata, respectively. These searches identified an additional 56 full copies of CMITEs from A. millepora, and 73 from A. palmata. Sequence analysis revealed that in comparison to CMITEs from families I, II, and III, all these elements had “shorter” TIRs in which the outermost regions matched their “partners” more closely than the innermost regions (Fig. 1). Since the FINDMITE program was mainly designed to identify MITEs with long and matching TIRs, this explained why most CMITEs had not been initially identified by that program. Based on the similarity of their TIRs, 113 of these elements (53 from A. millepora and 60 A. palmata) were classified into three additional families: families IV, V and VI (Table 1). Two of the remaining copies appeared to be degenerated copies of family III elements, and the other 14 were too degenerated to be unambiguously assigned to one of these families. Family IV was the largest of these families, outnumbering the others by a factor of two in A. palmata and almost by a factor of seven in A. millepora (Table 1). Within each family, there was no characteristic sequence difference between A. millepora and A. palmata elements, indicating that these families diverged prior to the coral species separation. Based on their observed frequency in A. millepora WGS sequences and the estimated genome size, we estimate the total number of CMITEs in the A. millepora genome at about 1600 copies. To further check for possible related elements in other species, we used the same query sequence to blast against the NCBI nr database, the Repbase database , and the WGS database for another coral species, Porites lobata. However, only one significant hit was found in the nr database, a partial lactate dehydrogenase (LDH)-like gene sequence (GenBank ID: EU814629) from A. millepora. A full copy of the CMITE element was located in the presumed intron region of this gene (data not shown).
An indication of a past transposition event of a CMITE was observed among A. millepora sequences, where we found two alleles of the same locus, one without a CMITE and another with the CMITE including the characteristic TSD, TTAA (Fig. 2a).
One of the characteristics of CMITEs is the TTAA target site duplication (TSD). To date, only one MITE superfamily, piggyBac/TTAA was known to be able to generate TTAA TSDs . This MITE superfamily was supposed to be dependent upon a superfamily of DNA transposon called piggyBac. Elements in the piggyBac superfamily generally have 12–19 bp TIRs containing a “CC[C/T]T” terminal motif, and generate TTAA TSDs . All the CMITEs we initially identified were consistent with these hallmarks of piggyBac transposons. When searched against the RepBase database , piggyBac-like sequences were also found in the WGS databases of A. millepora and A. palmata, and a recently released A. millepora larval transcriptome .
In order to investigate the relationship between CMITEs and piggyBac-like transposons, we decided to isolate piggyBac-like transposons from the A. millepora genome. Through direct and inverse PCR approaches, six piggyBac-like elements were isolated from the A. millepora genome (Table 2), and full-length sequences were obtained for four of them. All full-length piggyBac-like elements contained the hallmarks of typical piggyBac transposons. Partial sequences were obtained for the rest, of which one element has one TIR and a complete open reading frame (ORF), and another one has a complete ORF. Five of these were found in the A. millepora larval transcriptome, the expression of these elements during development strongly suggests the presence of functional piggyBac-like elements in the A. millepora genome.
Phylogenetic analysis of AmiPB1-6 and other piggyBac-like elements revealed five major clades (Fig. 3). Clades I, II, III and IV correspond to previously identified clades . Clade V is a new clade identified in this study. AmiPB1 to 6 are grouped in clade I, II and V, which suggests diverse origins of A. millepora piggyBac-like elements. Unexpectedly, AmiPB3 is grouped with NvePB1 from the sea anemone Nematostella vectensis rather than with other A. millepora elements in the same clade (Fig. 3, clade V). This may suggest that piggyBac clades diverged before the separation of the corresponding Cnidarian orders, Scleractinia and Actiniaria.
AmiPB1 was the only element isolated through the direct PCR approach. AmiPB1 and CMITE family I share identical TIR sequences, as well as weak sequence similarity in the internal region adjacent to TIRs (Fig. 2b). This suggests that family I is possibly the derivative of AmiPB1, and could utilize AmiPB1 transposase to mobilize in the genome. However, there is no obvious sequence similarity between the most of internal regions of CMITE family I and AmiPB1. For other piggyBac-like elements, except the hallmark terminal TIR motif (i.e., CC[C/T]T), which is necessary for successful transposition of piggyBac transposons , , we did not observe any obvious sequence similarities between these elements and CMITE families.
As mentioned above, 7 A. millepora and 12 A. palmata WGS sequences contained more than one CMITE element. Unexpectedly, some of these CMITE elements were found in tandem arrays, which typical MITEs usually do not form. Fig. 2c shows two examples of tandem CMITE arrays, including one with gaps between the repeated elements. Within these tandem arrays, both the elements themselves and the sequences between them are highly similar, implying that array formation was probably driven by a replication slippage mechanism rather than by independent transposition.
We also identified a CMITE-related family of elements (which we named CMITE-IN) in both A. millepora and A. palmata WGS sequences. CMITE-IN element contains a full copy of the CMITE element flanked by direct repeats, and has TTAA at both ends (examples: AM745001823 and AP824035187 in Fig. 2d). Four CMITE-IN elements were identified in the A. millepora WGS sequences, and one in the A. palmata WGS sequences. We estimate there are ~70 CMITE-IN copies in the A. millepora genome. A likely prototype of the CMITE-IN element was found in an A. palmata sequence (AP824030709, Fig. 2d), which contains two 23 bp direct repeats and has TTAA at both ends. The two direct repeats are separated by TTAA, which served as a target site for insertion of a CMITE element in the genome of another coral (Fig. 2d, top). The CMITE-IN element also seems to be a mobile element: we identified a pair of alleles from A. palmata, with and without CMITE-IN element, which suggest that the CMITE-IN can be excised at the position of its protoelement-derived TTAAs (Fig. 2d, bottom). We infer that the allele without the element (AP824033477) is a result of the past excision because it retains a possible TSD (Fig. 2d). Although piggyBac transposases usually perform precise excision without leaving “footprints” at the target sites , , imprecise excision events leaving TTAA TSDs in the target site were also observed . In the case presented here, one of these TSDs has apparently mutated into ACAA, possibly as a result of imperfect repair of the double-strand break after transposon excision.
To our knowledge, this is the first report of identification of MITEs from coral genomes. The CMITEs described here appear to have originated from piggyBac-like transposons. However, in comparison to other MITEs of the same origin –, CMITEs have the following noteworthy features:
The most unusual feature of CMITEs is conservation of the internal region, which is more conserved between MITE families than the TIRs. Typically, internal regions of different MITE families are much more dissimilar in size and sequence . In part, the conservation of the internal region in CMITEs may result from ascertainment bias, since the internal region was used as query to search for the majority of CMITEs. However, there was no such bias while initially detecting CMITEs using the FINDMITE program since it was based on TIR similarity only. It is tempting to speculate that the internal region is somehow important for CMITEs' transposition. Indeed, a recent study has shown that some internal sequences in MITEs could enhance transposition .
We showed that CMITE family I seems to be the derivative of a piggyBac-like transposon, AmiPB1. However, by comparison of CMITE family I and AmiPB1, we only observed very limited sequence similarity in the internal region adjacent to TIRs (Fig. 2b), and no obvious sequence similarity for the rest of internal region. Thus the origin of internal region of CMITEs remains a mystery. Interestingly, a recent study showed that host genomic sequences can be acquired by MITEs and filled in between TIRs through a process called transduplication . This could be a reasonable explanation for the origin of internal region of CMITEs. However, the WGS sequences currently available did not include any likely candidates for this putative original sequence, so complete genome sequences (which are unfortunately not yet available for any coral) will likely be required to resolve whether transduplication played a role in these elements.
In contrast to the internal region, TIRs between CMITE families are usually less conserved. However, all CMITE families preserved the terminal TIR motif (i.e., CC[C/T]T) (Table 1), which is a hallmark of TIRs of piggyBac transposons , and is necessary for successful transposition of piggyBac transposons , , so it is possible that this TIR motif coupled with the conserved internal region is already sufficient for successful transposition of these CMITE families. If this is the case, it might allow for cross-mobilization of these MITEs by various kinds of piggyBac-like transposons (Fig. 3), since TIRs of piggyBac-like transposons we identified in the A. millepora genome also preserved this motif (Table 2).
Our observations indicate that CMITEs can increase their copy numbers not only by transposition, but also by forming tandem arrays. To our knowledge, this is the first report of tandem arrays of full-sized MITEs, although tandem arrays formed by partial internal sequence of a piggyBac-MITE have been observed . Specific mechanisms responsible for the CMITEs array formation are unclear, but could be related to their similarity to the autonomous piggyBac transposons, which are also able to form large tandem arrays . Even if it is the case, however, CMITEs seem to be the only piggyBac-derived MITEs that retain this ability. This suggests that CMITEs contain some unique features that facilitate the formation of tandem arrays.
The finding of a novel TE family created by insertion of a CMITE suggests an unusual mechanism for the generation of novel TEs. Although we have shown the evidence of past mobility of the CMITE-IN element, the transposition mechanism remains unclear. CMITE-IN elements are structurally similar to miniature subterminal inverted-repeat transposable element (MSITE), which contain subterminal inverted-repeat (SIR) but no TIRs. Identification of MSITEs has been reported in several studies –. In one particular case, a 7 bp motif in the TIR of Wuneng (MITE) was found in the SIR of Microuli (MSITE) . Interestingly, both Wuneng and Microuli can generate TTAA TSDs. Based on these observations, the authors proposed that SIR might play an important role in MSITE transposition by providing key motifs. Since CMITE-IN and MSITE share similar TE structure, we speculate that the transposition mechanism of CMITE-IN may be also very similar to that of MSITE.
In summary, we present the first report of non-autonomous MITE-like elements (CMITEs) from two coral genomes. These elements bear the telltale features of MITEs related to piggyBac-like autonomous transposons. We show that the coral genome indeed contains such autonomous transposons, most of which are also transcriptional active, ostensibly providing the transposition machinery for the CMITEs. The unusually well-conserved internal region of CMITEs suggests a potentially important role in successful transposition. However, the origin of these unusual features in CMITEs remains unclear, and represents an intriguing topic for future studies.
AmiPB1-6 and other piggyBac-like transposase protein sequences (fasta and ‘aligned’ formats).
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Competing Interests: The authors have declared that no competing interests exist.
Funding: The authors have no support or funding to report.