Transduplication resulted in the incorporation of two protein-coding sequences into the Turmoil-1 transposable element of C. elegans

Transposable elements may acquire unrelated gene fragments into their sequences in a process called transduplication. Transduplication of protein-coding genes is common in plants, but is unknown of in animals. Here, we report that the Turmoil-1 transposable element in C. elegans has incorporated two protein-coding sequences into its inverted terminal repeat (ITR) sequences. The ITRs of Turmoil-1 contain a conserved RNA recognition motif (RRM) that originated from the rsp-2 gene and a fragment from the protein-coding region of the cpg-3 gene. We further report that an open reading frame specific to C. elegans may have been created as a result of a Turmoil-1 insertion. Mutations at the 5' splice site of this open reading frame may have reactivated the transduplicated RRM motif. This article was reviewed by Dan Graur and William Martin. For the full reviews, please go to the Reviewers' Reports section.


Findings
The possible contribution of transposable elements to the proteome has been discussed in several publications [1][2][3][4][5][6][7] and has provoked much debate [8]. Many mechanisms are known to increase the protein-domain repertoire, e.g., domain duplication, substitution mutations, insertions, deletions, and domain rearrangements [9]. In metazoans, a transposable element may result in transduction, in which a DNA segment downstream of transposable elements is mobilized as part of an aberrant transposition. This may result in gene duplication or exon shuffling, subsequently enriching the protein repertoire [10][11][12][13]. How-ever, in the process of transduction, the transposable element does not acquire gene fragments as part of its sequence.
In plants, on the other hand, thousands of transposable elements contain duplicated gene fragments, captured in a process termed transduplication. Transduplication is a potentially rich source of novel coding sequences within rice and Arabidopsis thaliana [14][15][16]. Recently, transduplications of small nucleolar (sno) RNA by retroposon-like non-LTR transposable elements were found in the C. elegans [17] and platypus genomes [18].
The Harbinger superfamily of "cut-and-paste" DNA transposons was discovered through in silico studies [19]. This superfamily is characterized by Harbinger-specific transposases that are distantly related to the transposases encoded by the IS5-like group of bacterial transposons, such as IS5, IS112, and ISL2. Harbinger transposons are not as widespread as the eukaryotic hAT and mariner/Tc1 transposons; they are found in plants and nematodes [19][20][21][22][23] but not in mammals. Usually, Harbinger transposons are flanked by 3-bp target site duplications and 25-to 50bp inverted terminal repeats (ITRs).
Turmoil-1 is a 5,024-bp long DNA transposon with 760bp long ITRs and a Harbinger-specific transposase ( Figure  1A). These ITRs are unique to Turmoil-1 and are not found in other members of the Harbinger superfamily of DNA transposons [24]. One complete copy of the Turmoil-1 was found on chromosome II of C. elegans; eight Turmoil-1 fragments exist in the genome (for detailed information, see Table 1). An analysis of C. elegans transposable elements [25,26] revealed that a 205-bp ITR sequence within Turmoil-1 is highly similar to a region of two exons separated by an intron of the rsp-2 gene (see pairwise alignment using bl2seq [27] Figure 1B). These two exons encode the RNA Recognition Motif (RRM), which is found in many eukaryal and bacterial proteins. Specifically, the type of RRM domain present in the rsp-2 (called RRM1) gene is highly conserved evolutionarily [28]. The high similarity between the ITR sequence and the fragment of the rsp-2 gene implies that one originated from the other. The antiquity of this domain and a phyloge-netic analysis ( Figure 2) indicate that Turmoil-1 has recently acquired a portion of the rsp-2 gene sequence into its ITR. Tree reconstruction was performed with the PhyML program version 2.4.5 [29] using among-site rate variation with four discrete rate categories, and the JTT model [30] of sequence evolution.
A comparative analysis of the rsp-2 gene and the 205-bp region of the gene found in the ITR sequence, revealed that the Turmoil-1 sequence has accumulated several point mutations within the 5' splice site that make it non-functional, whereas the 3' splice site of the intron remains intact (the mutations in the 5' splice site region are marked in red in Figure 1B). Since the RRM domain within the Turmoil-1 DNA transposon is not under purifying selection to maintain the reading frame or the functionality of the splice sites, these mutations are not unexpected.
Within the same ITR domain of Turmoil-1, and very close to the site of insertion of the RRM domain of rsp-2 gene, there is evidence of another "DNA kidnapping" event. A 131-bp fragment from the coding region of C. elegans gene cpg-3, which is unique to nematodes, was inserted into the ITR ( Figure 1C). No sequences homologous to Turmoil-1 flank the cpg-3 gene. Thus, similar to the rsp-2 case, a gene fragment from the cpg-3 most likely was acquired by Turmoil-1, and not vice-versa. As the gene fragments are present on both sides of the ITR, capture may have occurred through non-homologous recombination.  Evidence for two transduplication events within the ITR sequence of the Turmoil-1 transposon  (see table 1, copy number 6) and the structure and sites of inclusion of fragments of the rsp-2 and cpg-3 genes within the ITR sequence (B) Pairwise alignment (using bl2seq [27]) between the Turmoil-1 sequence and the rsp-2 sequence. The start codon, which is also the first amino acid of the RRM1 domain in the rsp-2 gene, is boxed in red. The intron sequence is indicated by lower-case letters; the protein-coding region is indicated by upper-case letters. The positions of the 5' and 3' splice sites (5'ss and 3'ss, respectively) are marked with arrows. The nucleotides in the 5' splice site of the rsp-2 gene that were mutated in the sequence of the transposable element, thereby abolishing the original 5' splice site, are shown in red. (C) Pairwise alignment between Turmoil-1 and the cpg-3 sequence.
Maximum likelihood tree of the RRM domain Figure 2 Maximum likelihood tree of the RRM domain. The RRM domain sequence, which is part of the ITR of Turmoil-1, is indicated in red. The tree shows that the RRM domain within Turmoil-1 is derived from the ancestral RRM domain rather than vice versa. One of the Turmoil-1 copies (number 1 in Table 1) contains within it an open reading frame (ORF) with the accession number Y48G1BL.4. It contains two putative exons and an intron (Figure 3), which are similar to the RRM domain. The 5' splice site that corresponds to that in the rsp-2 transcript has been mutated. A novel 5' splice site is most likely located nine nucleotides downstream from the original one. At this site, a point mutation changed an AT into a GT dinucleotide (marked in red in Figure 3). Usage of this 5' splice site maintains the ORF equivalent to that of the RRM domain of rsp-2 with the exception of the addition of three amino acids. These additional residues should have a negligible effect on the three-dimensional structure of the RRM domain ( Figure 3C). This ORF, however, may not be transcriptionally active as its sequence is only found in the UNIPROT database (accession number Q9N3P9), and there is no EST or cDNA supporting evidence. If this is the case, it would be consistent with reports that indicate that all known transduplicates in rice, in spite of their genomic abundance, are pseudogenes [16].

Y48G1BL.4 gene with predicted RRM domain
Our analysis indicates that Turmoil-1 of C. elegans has captured two unrelated coding sequences within its ITRs at proximate locations. The presence of a transduplication "hotspot" in this region may be tentatively inferred. This analysis reveals a transduplication of protein-coding regions in C. elegans and strengthens the hypothesis that protein domains may be mobilized by transposable elements.