Landscape and evolutionary dynamics of Helitron transposons in plant genomes as well as construction of online database HelDB

Helitron transposons play an important role in host genome evolution due to their ability to capture genes and regulatory elements. In this study, we developed a pipeline to identify and annotate Helitrons systematically from 358 plant and 178 animal high-quality genomes. All these data were organized into HelDB, a database where Helitrons can be explored with a user-friendly Web interface and related software. Based on these data, further analysis showed that the number or the cumulative length of Helitrons is positively correlated with genome size. Helitrons had experienced two expansion periods in plants, with the first occurring 20–30 Ma and peaking at approximately 24 Ma. The second expansion occurred in the last 4 million years. The expansions might be due to stimulation of paleogeographic environment. Detailed investigation of gene capture by Helitrons in Brassicaceae and Solanaceae plants showed that the captured genes showed diverse functions. Interestingly, metal ion binding function was enriched in these captured genes in most species. This phenomenon might be due to the need for binding of divalent metal ions to the Rep domain required for Helitron transposition. This study improves our knowledge of the landscape and evolution of Helitron transposons in plants and paves a way for further functional studies of this kind of transposable element.     

Helitrons on a roll: eukaryotic rolling-circle transposons

Rolling-circle eukaryotic transposons, known as Helitron transposons, were first discovered in plants (Arabidopsis thaliana and Oryza sativa) and in the nematode Caenorhabditis elegans. To date, Helitrons have been identified in a diverse range of species, from protists to mammals. They represent a major class of eukaryotic transposons and are fundamentally different from classical transposons in terms of their structure and mechanism of transposition. Helitrons seem to have a major role in the evolution of host genomes. They frequently capture diverse host genes, some of which can evolve into novel host genes or become essential for helitron transposition.     

Mobilizing the genome of Lepidoptera through novel sequence gains and end creation by non-autonomous Lep1 Helitrons

Transposable elements (TEs) can affect the structure of genomes through their acquisition and transposition of novel DNA sequences. The 134-bp repetitive elements, Lep1, are conserved non-autonomous Helitrons in lepidopteran genomes that have characteristic 5'-CT and 3'-CTAY nucleotide termini, a 3'-terminal hairpin structure, a 5'- and 3'-subterminal inverted repeat (SIR), and integrations that occur between AT or TT nucleotides. Lep1 Helitrons have acquired and propagated sequences downstream of their 3'-CTAY termini that are 57-344-bp in length and have termini composed of a 3'-CTRR preceded by a 3'-hairpin structure and a region complementary to the 5'-SIR (3'-SIRb). Features of both the Lep1 Helitron and multiple acquired sequences indicate that secondary structures at the 3'-terminus may have a role in rolling circle replication or genome integration mechanisms, and are a prerequisite for novel end creation by Helitron-like TEs. The preferential integration of Lep1 Helitrons in proximity to gene-coding regions results in the creation of genetic novelty that is shown to impact gene structure and function through the introduction of novel exon sequence (exon shuffling). These findings are important in understanding the structural requirements of genomic DNA sequences that are acquired and transposed by Helitron-like TEs.     

Tdd-4, a DNA transposon of Dictyostelium that encodes proteins similar to LTR retroelement integrases.

Tdd-4 is the first DNA transposon to be isolated from Dictyostelium discoideum. This element was isolated by insertion into a target plasmid. Two classes of elements were identified which include a 3.8 kb version and a 3.4 kb deleted version. Sequence analysis reveals that the 145 bp inverted terminal repeats contain the 5'-TGellipsisCA-3' conserved terminal dinucleotides found in prokaryotic transposons and integrated LTR retroelement DNA sequences. Tdd-4 open reading frames are assembled by removal of six introns. Introns 1-5 conform to the GT-AG rule, whereas intron 6 appears to be an AT-AA intron. Also, intron 6 undergoes an alternative 5' splicing reaction. The alternatively spliced region encodes 15 tandem SPXX repeats that are proposed to function as a DNA binding motif. By analogy to other transposons that encode two proteins from the same gene, the full-length Tdd-4 protein is the putative transposase and the truncated Tdd-4 protein is the putative transposition inhibitor. Protein database searches demonstrate Tdd-4 encoded proteins are unique for a DNA element by containing similarities to retroviral/retrotransposon integrases. The putative Tdd-4 transposase contains the same structural relationship as integrases by possessing an N-terminal HHCC motif, a central DDE motif and a C-terminal DNA-binding domain composed of the SPXX motif.     

尖孢镰刀菌FoHeli转座元件的结构特征及功能验证

Helitron是一种广泛存在于真核生物中的可移动遗传元件。与其他转座子不同,自主Helitron元件可编码具有复制引发（Rep）和解旋酶（Hel）结构域的转座酶,并通过滚环复制的方式在基因组中进行扩张。本研究对9种尖孢镰刀菌中的自主Helitron元件进行系统分析,结果表明尖孢镰刀菌中存在两类自主Helitron元件FoHeli1与FoHeli2。其中FoHeli1成员间序列高度相似,并具有明晰的边界特征：3’端为保守的“TATTTT”序列,其上游可形成稳定的发夹结构,且发夹上游可与5’端形成12bp的反向互补结构。基于上述分析结果,从尖孢镰刀菌Fo4287菌株中克隆获得完整的FoHeli1元件,并通过构建双元转座系统及PEG介导的原生质体转化,证明尖孢镰刀菌中的FoHeli元件可在禾谷镰刀菌PH-1菌株的基因组中发生跳转。     

Identification of active transposon dTok, a member of the hAT family, in rice

Recent completion of the sequencing of the rice genome has revealed that it contains >40% repetitive sequences, most of which are related to inactive transposable elements. During the molecular analysis of the floral organ number 1/multiple pistil 2 (fon1/mp2) mutant, we identified an active transposable element dTok0 that was inserted at the kinase domain of FON1, a homolog of CLAVATA1. Insertion of the element into FON1 generated an 8 bp duplication of its target sites, which is one of the major characteristics of the hAT family of transposons. The dTok0 element was actively transposed out of the FON1 gene, leaving 5-8 bp footprints. Reinsertion into a new location was observed at a low frequency. Analysis of the genome sequence showed that the rice cultivar 'Nipponbare' contains 25 copies of dTok elements; similar numbers were present in all the Oryza species examined. Because dTok0 does not encode a transposase, enzyme activity should be provided in trans. We identified a putative autonomous transposon, Tok1 that contains an intact open reading frame of the Ac-like transposase.     

Model-based identification of Helitrons results in a new classification of their families in Arabidopsis thaliana

Helitrons are a class of prolific transposable elements in the Arabidopsis thaliana genome. Although 37 families were identified after the recent discovery of Helitrons, no systematic classification is available because of the high variability of helitronic sequences. Since transposition proteins are assumed to interact with Helitron termini, a Helitron model was formalized based on terminus characterization in order to carry out an exhaustive analysis of all possible combinations of the pairs of termini present. This combinatorics approach resulted in the discovery of a number of new Helitron elements corresponding to termini associations from distinct previously-described Helitron families. The occurrence matrix of termini combinations yielded a structure that revealed clusters of Helitron families.     

MMTS, a new subfamily of Tc1-like transposons

A novel Tc1-like transposable element has been identified as a new DNA transposon in the mud loach, Misgurnus mizolepis. The M. mizolepis Tc1-like transposon (MMTS) is comprised of inverted terminal repeats and a single gene that codes Tc1-like transposase. The deduced amino acid sequence of the transposase-encoding region of MMTS transposon contains motifs including DDE motif, which was previously recognized in other Tc1-like transposons. However, putative MMTS transposase has only 34-37% identity with well-known Tc1, PPTN, and S elements at the amino acid level. In dot-hybridization analysis used to measure the copy numbers of the MMTS transposon in genomes of the mud loach, it was shown that the MMTS transposon is present at about 3.36 x 104 copies per 2 x 109 bp, and accounts for approximately 0.027% of the mud loach genome. Here, we also describe novel MMTS-like transposons from the genomes of carp-like fishes, flatfish species, and cichlid fishes, which bear conserved inverted repeats flanking an apparently intact transposase gene. Additionally, BLAST searches and phylogenetic analysis indicated that MMTS-like transposons evolved uniquely in fishes, and comprise a new subfamily of Tc1-like transposons, with only modest similarity to Drosophila melanogaster (foldback element FB4, HB2, HB1), Xenopus laevis, Xenopus tropicalis, and Anopheles gambiae (Frisky).     

<Emphasis Type="Italic">Helitron</Emphasis> mediated amplification of cytochrome P450 monooxygenase gene in maize

The mass movement of gene sequences by Helitrons has significantly contributed to the lack of gene collinearity reported between different maize inbred lines. However, Helitron captured-genes reported to date represent truncated versions of their progenitor genes. In this report, we provide evidence that maize CYP72A27-Zm gene represents a cytochrome P450 monooxygenase (P450) gene recently captured by a Helitron and transposed into an Opie-2 retroposon. The four exons of the CYP72A27 gene contained within the element contain a putative open reading frame (ORF) for 428 amino acid residues. We provide evidence that Helitron captured CYP72A27-Zm is transcribed. To identify the progenitor gene and the evolutionary time of capture, we searched the plant genome database and discovered other closely related CYP72A27-Zm genes in maize and grasses. Our analysis indicates that CYP72A27-Zm represents an almost complete copy of maize CYP72A26-Zm gene captured by a Helitron about 3.1 million years ago (mya). The Helitron-captured gene then duplicated twice, approximately 1.5-1.6 mya giving rise to CYP72A36-Zm and CYP72A37-Zm. These data provide evidence that Helitrons can capture and mobilize intact genes that are transcribed and potentially encode biologically relevant proteins.     

Molecular characterization of a mariner-like element in the Atta sexdens rubropilosa genome

Mobile elements are widely present in eukaryotic genomes. They are repeated DNA segments that are able to move from one locus to another within the genome. They are divided into two main categories, depending on their mechanism of transposition, involving RNA (class I) or DNA (class II) molecules. The mariner-like elements are class II transposons. They encode their own transposase, which is necessary and sufficient for transposition in the absence of host factors. They are flanked by a short inverted terminal repeat and a TA dinucleotide target site, which is duplicated upon insertion. The transposase consists of two domains, an N-terminal inverted terminal repeat binding domain and a C-terminal catalytic domain. We identified a transposable element with molecular characteristics of a mariner-like element in Atta sexdens rubropilosa genome. Identification started from a PCR with degenerate primers and queen genomic DNA templates, with which it was possible to amplify a fragment with mariner transposable-element homology. Phylogenetic analysis demonstrated that this element belongs to the mauritiana subfamily of mariner-like elements and it was named Asmar1. We found that Asmar1 is homologous to a transposon described from another ant, Messor bouvieri. The predicted transposase sequence demonstrated that Asmar1 has a truncated transposase ORF. This study is part of a molecular characterization of mobile elements in the Atta spp genome. Our finding of mariner-like elements in all castes of this ant could be useful to help understand the dynamics of mariner-like element distribution in the Hymenoptera.     

A purified mariner transposase is sufficient to mediate transposition in vitro.

Mariners are a widespread and diverse family of animal transposons. Extremely similar mariners of the irritans subfamily are present in the genomes of three divergent insect host species, which strongly suggests that species-specific host factors are unnecessary for mobility. We tested this hypothesis by examining the activity of a purified transposase from one of these elements (Himar1) present in the horn fly, Haematobia irritans. Himar1 transposase was sufficient to reproduce transposition faithfully in an in vitro inter-plasmid transposition reaction. Further analyses showed that Himar1 transposase binds to the inverted terminal repeat sequences of its cognate transposon and mediates 5' and 3' cleavage of the element termini. Independence of species-specific host factors helps to explain why mariners have such a broad distribution and why they are capable of horizontal transfer between species.     

Retargeting transposon insertions by the adeno-associated virus Rep protein

The Sleeping Beauty (SB), piggyBac (PB) and Tol2 transposons are promising instruments for genome engineering. Integration site profiling of SB, PB and Tol2 in human cells showed that PB and Tol2 insertions were enriched in genes, whereas SB insertions were randomly distributed. We aimed to introduce a bias into the target site selection properties of the transposon systems by taking advantage of the locus-specific integration system of adeno-associated virus (AAV). The AAV Rep protein binds to Rep recognition sequences (RRSs) in the human genome, and mediates viral integration into nearby sites. A series of fusion constructs consisting of the N-terminal DNA-binding domain of Rep and the transposases or the N57 domain of SB were generated. A plasmid-based transposition assay showed that Rep/SB yielded a 15-fold enrichment of transposition at a particular site near a targeted RRS. Genome-wide insertion site analysis indicated that an approach based on interactions between the SB transposase and Rep/N57 enriched transgene insertions at RRSs. We also provide evidence of biased insertion of the PB and Tol2 transposons. This study provides a comparative insight into target site selection properties of transposons, as well as proof-of-principle for targeted chromosomal transposition by composite protein-protein and protein-DNA interactions.     

Horizontal Escape of the Novel Tc1-Like Lepidopteran Transposon TCp3.2 into Cydia pomonella Granulovirus

We characterized an insertion mutant of the baculovirus Cydia pomonella granulovirus (CpGV), which contained a transposable element of 3.2 kb. This transposon, termed TCp3.2, has unusually long inverted terminal repeats (ITRs) of 756 bp and encodes a defective gene for a putative transposase. Amino acid sequence comparison of the defective transposase gene revealed a distant relationship to a putative transposon in Caenorhabditis elegans which also shares some similarity of the ITRs. Maximum parsimony analysis of the predicted amino acid sequences of Tc1- and mariner-like transposases available from the GenBank data base grouped TCp3.2 within the superfamily of Tc1-like transposons. DNA hybridization indicated that TCp3.2 originated from the genome of Cydia pomonella, which is the natural host of CpGV, and is present in less than 10 copies in the C. pomonella genome. The transposon TCp3.2 most likely was inserted into the viral genome during infection of host larvae. TCp3.2 and the recently characterized Tc1-like transposon TC14.7 (Jehle et al. 1995), which was also found in a CpGV mutant, represent a new family of transposons found in baculovirus genomes. The occasional horizontal escape of different types of host transposons into baculovirus genomes evokes the question about the possible role of baculoviruses as an interspecies vector in the horizontal transmission of insect transposons. Received: 27 February 1997 / Accepted: 16 May 1997     

Pif1 Helicases and the Evidence for a Prokaryotic Origin of Helitrons

Helitrons are the only group of rolling-circle transposons that encode a transposase with a helicase domain (Hel), which belongs to the Pif1 family. Because Pif1 helicases are important components of eukaryotic genomes, it has been suggested that Hel domains probably originated after a host eukaryotic Pif1 gene was captured by a Helitron ancestor. However, the few analyses exploring the evolution of Helitron transposases (RepHel) have focused on its Rep domain, which is also present in other mobile genetic elements. Here, we used phylogenetic and nonmetric multidimensional scaling analyses to investigate the relationship between Hel domains and Pif1-like helicases from a variety of organisms. Our results reveal that Hel domains are only distantly related to genomic helicases from eukaryotes and prokaryotes, and thus are unlikely to have originated from a captured Pif1 gene. Based on this evidence, and on recent studies indicating that Rep domains are more closely related to rolling-circle plasmids and phages, we suggest that Helitrons are descendants of a RepHel-encoding prokaryotic plasmid element that invaded eukaryotic genomes before the radiation of its major groups. We discuss how a Pif1-like helicase domain might have favored the transposition of Helitrons in eukaryotes beyond simply unwinding DNA intermediates. Finally, we demonstrate that some examples in the literature describing genomic helicases from eukaryotes actually consist of Hel domains from Helitrons, a finding that underscores how transposons can hamper the analysis of eukaryotic genes. This investigation also revealed that two groups of land plants appear to have lost genomic Pif1 helicases independently.     

The proteins encoded by the pogo-like Lemi1 element bind the TIRs and subterminal repeated motifs of the Arabidopsis Emigrant MITE: consequences for the transposition mechanism of MITEs

MITEs (miniature inverted-repeated transposable elements) are a particular class of defective DNA transposons usually present within genomes as high copy number populations of highly homogeneous elements. Although an active MITE, the mPing element, has recently been characterized in rice, the transposition mechanism of MITEs remains unknown. It has been proposed that transposases of related transposons could mobilize MITEs in trans. Moreover, it has also been proposed that the presence of conserved terminal inverted-repeated (TIR) sequences could be the only requirement of MITEs for mobilization, allowing divergent or unrelated elements to be mobilized by a particular transposase. We present here evidence for a recent mobility of the Arabidopsis Emigrant MITE and we report on the capacity of the proteins encoded by the related Lemi1 transposon, a pogo-related element, to specifically bind Emigrant elements. This suggests that Lemi1 could mobilize Emigrant elements and makes the Lemi1/Emigrant couple an ideal system to study the transposition mechanism of MITEs. Our results show that Lemi1 proteins bind Emigrant TIRs but also bind cooperatively to subterminal repeated motifs. The requirement of internal sequences for the formation of proper DNA/protein structure could affect the capacity of divergent MITEs to be mobilized by distantly related transposases.     

Activation and epigenetic regulation of DNA transposon nDart1 in rice

A large part of the rice genome is composed of transposons. Since active excision/reintegration of these mobile elements may result in harmful genetic changes, many transposons are maintained in a genetically or epigenetically inactivated state. However, some non-autonomous DNA transposons of the nDart1-3 subgroup, including nDart1-0, actively transpose in specific rice lines, such as pyl-v which carries an active autonomous element, aDart1-27, on chromosome 6. Although nDart1-3 subgroup elements show considerable sequence identity, they display different excision frequencies. The most active element, nDart1-0, had a low cytosine methylation status. The aDart1-27 sequence showed conservation between pyl-stb (pyl-v derivative line) and Nipponbare, which both lack autonomous activity for transposition of nDart1-3 subgroup elements. In pyl-v plants, the promoter region of the aDart1-27 transposase gene was more hypomethylated than in other rice lines. Treatment with the methylation inhibitor 5-azacytidine (5-azaC) induced transposition of nDart1-3 subgroup elements in both pyl-stb and Nipponbare plants; the new insertion sites were frequently located in genic regions. 5-AzaC treatment principally induced expression of Dart1-34 transposase rather than the other 38 aDart1-related elements in both pyl-stb and Nipponbare treatment groups. Our observations show that transposition of nDart1-3 subgroup elements in the nDart1/aDart1 tagging system is correlated with the level of DNA methylation. Our system does not cause somaclonal variation due to an absence of transformed plants, offers the possibility of large-scale screening in the field and can identify dominant mutants. We therefore propose that this tagging system provides a valuable addition to the tools available for rice functional genomics.     

A family of Tc1-like transposons from the genomes of fishes and frogs: evidence for horizontal transmission.

Tc1-like transposons are very widely distributed within the genomes of animal species. They consist of an inverted repeat sequence flanking a transposase gene with homology to the mobile DNA element, Tc1 of the nematode Caenorhabditis elegans. These elements seem particularly to infest the genomes of fish and amphibian species where they can account for 1% of the total genome. However, all vertebrate Tc1-like elements isolated so far are non-functional in that they contain multiple frameshifts within their transposase coding regions. Here I describe a Tc1-like transposon (PPTN) from the genome of a marine flatfish species (Pleuronectes platessa) which bears conserved inverted repeats flanking an apparently intact transposase gene. Closely related, although degenerate, Tc1-like transposons were also isolated from the genomes of Atlantic salmon (SSTN, Salmo salar) and frog (RTTN, Rana temporaria). Consensual nucleic acid sequences were derived by comparing several individual isolates from each species and conceptual amino acid sequences were thence derived for their transposases. Phylogenetic analysis of these sequences with previously isolated Tc1-like transposases shows that the elements from plaice, salmon and frog comprise a new subfamily of Tc1-like transposons. Each member is distinct in that it is not found in the genomes of the other species tested. Plaice genomes contain about 300 copies of PPTN, salmon 1200 copies of SSTN and frog genomes about 500 copies of RTTN. The presence of these closely related elements in the genomes of fish and frog species, representing evolutionary lines, which diverged more than 400 million years ago, is not consistent with a vertical transmission model for their distributions.     

Regulation of mariner transposition: the peculiar case of mos1

Background

Mariner elements represent the most successful family of autonomous DNA transposons, being present in various plant and animal genomes, including humans. The introduction and co-evolution of mariners within host genomes imply a strict regulation of the transposon activity. Biochemical data accumulated during the past decade have led to a convergent picture of the transposition cycle of mariner elements, suggesting that mariner transposition does not rely on host-specific factors. This model does not account for differences of transposition efficiency in human cells between mariners. We thus wondered whether apparent similarities in transposition cycle could hide differences in the intrinsic parameters that control mariner transposition.

Principal Findings

We find that Mos1 transposase concentrations in excess to the Mos1 ends prevent the paired-end complex assembly. However, we observe that Mos1 transposition is not impaired by transposase high concentration, dismissing the idea that transposase over production plays an obligatory role in the down-regulation of mariner transposition. Our main finding is that the paired-end complex is formed in a cooperative way, regardless of the transposase concentration. We also show that an element framed by two identical ITRs (Inverted Terminal Repeats) is more efficient in driving transposition than an element framed by two different ITRs (i.e. the natural Mos1 copy), the latter being more sensitive to transposase concentration variations. Finally, we show that the current Mos1 ITRs correspond to the ancestral ones.

Conclusions

We provide new insights on intrinsic properties supporting the self-regulation of the Mos1 element. These properties (transposase specific activity, aggregation, ITR sequences, transposase concentration/transposon copy number ratio…) could have played a role in the dynamics of host-genomes invasion by Mos1, accounting (at least in part) for the current low copy number of Mos1 within host genomes.