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. 2020 Dec 10;11(1):32.
doi: 10.1186/s13100-020-00227-7.

Intruder (DD38E), a recently evolved sibling family of DD34E/Tc1 transposons in animals

Bo Gao  1   2 , Wencheng Zong  1 , Csaba Miskey  2 , Numan Ullah  1 , Mohamed Diaby  1 , Cai Chen  1 , Xiaoyan Wang  1 , Zoltán Ivics  2 , Chengyi Song  3
Affiliations

Intruder (DD38E), a recently evolved sibling family of DD34E/Tc1 transposons in animals

Bo Gao et al. Mob DNA. .

Abstract

Background: A family of Tc1/mariner transposons with a characteristic DD38E triad of catalytic amino acid residues, named Intruder (IT), was previously discovered in sturgeon genomes, but their evolutionary landscapes remain largely unknown.

Results: Here, we comprehensively investigated the evolutionary profiles of ITs, and evaluated their cut-and-paste activities in cells. ITs exhibited a narrow taxonomic distribution pattern in the animal kingdom, with invasions into two invertebrate phyla (Arthropoda and Cnidaria) and three vertebrate lineages (Actinopterygii, Agnatha, and Anura): very similar to that of the DD36E/IC family. Some animal orders and species seem to be more hospitable to Tc1/mariner transposons, one order of Amphibia and seven Actinopterygian orders are the most common orders with horizontal transfer events and have been invaded by all four families (DD38E/IT, DD35E/TR, DD36E/IC and DD37E/TRT) of Tc1/mariner transposons, and eight Actinopterygii species were identified as the major hosts of these families. Intact ITs have a total length of 1.5-1.7 kb containing a transposase gene flanked by terminal inverted repeats (TIRs). The phylogenetic tree and sequence identity showed that IT transposases were most closely related to DD34E/Tc1. ITs have been involved in multiple events of horizontal transfer in vertebrates and have invaded most lineages recently (< 5 million years ago) based on insertion age analysis. Accordingly, ITs presented high average sequence identity (86-95%) across most vertebrate species, suggesting that some are putatively active. ITs can transpose in human HeLa cells, and the transposition efficiency of consensus TIRs was higher than that of the TIRs of natural isolates.

Conclusions: We conclude that DD38E/IT originated from DD34E/Tc1 and can be detected in two invertebrate phyla (Arthropoda and Cnidaria), and in three vertebrate lineages (Actinopterygii, Agnatha and Anura). IT has experienced multiple HT events in animals, dominated by recent amplifications in most species and has high identity among vertebrate taxa. Our reconstructed IT transposon vector designed according to the sequence from the "cat" genome showed high cut-and-paste activity. The data suggest that IT has been acquired recently and is active in many species. This study is meaningful for understanding the evolution of the Tc1/mariner superfamily members and their hosts.

Keywords: DD38E; Evolution; Horizontal transfer; Intruder; Tc1/mariner transposons.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Taxonomic distribution of DD38E/IT. a Taxonomic distribution of IT elements in the animal kingdom. N represents the number of species with IT. b Description of IT elements in six lineages including the number of species with these elements, full length (FL) of the elements, amino acid (aa) numbers of transposases (TPase), lengths of terminal inverse repeats (TIRs) and target site duplications (TSDs)
Fig. 2
Fig. 2
Distribution patterns of DD38E/IT, DD35E/TR, DD36E/IC and DD37E/TRT. a Distribution of IT, TR, IC and TRT transposons. The numbers of species/orders detected for each family are indicated for each lineage. b, c Venn diagrams of distribution patterns across orders and species. Figures were generated from the Supplementary File Text S3
Fig. 3
Fig. 3
Phylogenetic position of the IT family. a This phylogenetic tree was generated based on DDE domains by using the Maximum Likelihood method in the IQ-TREE program (http://iqtree.cibiv.univie.ac.at) with an ultrafast bootstrap approach (1000 replicates). The reference families and elements included DD34E/Tc1, DD35E/TR, DD36E/IC, DD37E/TRT, DD34D/mariner, DD37D/maT, DD39D, DD41D/VS, DD × D/pogo and IS630 transposases. TP36/Zator was used as an outgroup. b Sequence identity matrix of Tc1/mariner families by pairwise comparisons among FL transposases
Fig. 4
Fig. 4
Structural schematic of IT transposons. a Structural organization of IT elements. The green arrows represent TIRs, the black rectangles represent HTH motifs, the black triangles represent GRPR sequences, the yellow circle represents the NLS, the orange rectangles represent catalytic domains, and the grey regions represent transposases. The dotted box represents the portion of the transposases that might be deleted in a particular species. b IT in the “cat” genome. The grey areas at the top and bottom represent IT. We selected copies 1 and 2 to mark the locations in this genome. In the middle is a schematic diagram of the complete IT structure. The red arrows represent TIRs, the green rectangle represents the DNA-binding domain, and the orange rectangle represents the catalytic domain. c Motifs prediction for IT transposases. This analysis was performed using multiple alignment with Bioedit and with modifications in Genedoc. Species abbreviations: Cafl, Camponotus floridanus; Danaus plexippus plexippus; Hyvu, Hydra vulgaris; Rhma, Rhinella marina; Sedu, Seriola dumerili; Stpa, Stegastes partitus
Fig. 5
Fig. 5
HT analysis of IT transposons. a Phylogenetic tree based on alignment of the nucleotide sequence of IT transposons. The phylogenetic tree was inferred using the maximum likelihood method with the IQ-TREE program (http://iqtree.cibiv.univie.ac.at), and the DD34E/Tc1 family was used as the outgroup, the identity calculation of each cluster was done using MEGA7. Only consensus or representative sequences were used in this analysis. b Horizontal transfer of IT transposons. The distance was obtained from all possible pairwise comparisons (n = 629; marked on the x-axis) between the 35 (cluster 2), five (cluster 3), three (cluster 4) and seven (cluster 5) species in which IT motifs were identified and complete. The coding sequence (CDS) of the RAG1 gene from the NCBI database is available in Supplementary Table S2
Fig. 6
Fig. 6
Insertion ages of ITs. This taxonomic tree represents the distribution of the species identified in the animal kingdom, and each colour represents a phylogenetic tree cluster. Insert age analysis was calculated by using the RepeatMasker program. The phylogenetic relationships were taken from the TimeTree database
Fig. 7
Fig. 7
IT is transpositionally active in mammalian cells. a IT and Sleeping Beauty (SB) transposon vectors for in vitro transposition activity assays. SB was used as a positive control. The three TIR vectors—pITo-Neo, pITc-Neo and pSB-Neo—have the same vector frame, and the TIR elements on both sides are the only differences. pITo-Neo is composed of two original TIR sequences of IT, pITc-Neo is composed of one consistent TIR and the other original TIR of IT, and pSB-Neo is composed of two SB TIRs. The two transposase vectors pCMV-itTPase and pCMV-SB100X also have the same frame. b–e HeLa cells were stably transfected with 1 μg of mixed plasmids (donor and helper plasmids at a 1:1 ratio. For selection, the transfected cells were reseeded onto 10-cm dishes (b and c, 1:100 plating; d and e, 1:10 plating). Selection was performed using 1000 mg/ml G418 for 14 days in DMEM. To determine the relative rates of transgenesis, foci of neomycin-resistant cell colonies that remained on each dish were counted after being fixed in 4% paraformaldehyde and stained with methylene blue. Bars represent the mean neomycin-resistant colonies ± standard deviations from three independent experiments
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