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R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, precise RNA-mediated insertion of transgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

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A single strain of Candida anglica, isolated from cider, is available in international yeast collections. We present here seven new strains isolated from French PDO cheeses. For one of the cheese strains, we achieved a high-quality genome assembly of 13.7 Mb with eight near-complete telomere-to-telomere chromosomes. The genomes of two additional cheese strains and of the cider strain were also assembled and annotated, resulting in a core genome of 5966 coding sequences. Phylogenetic analysis showed that the seven cheese strains clustered together, away from the cider strain. Mating-type locus analysis revealed the presence of a MATa locus in the cider strain but a MATalpha locus in all cheese strains. The presence of LINE retrotransposons at identical genome position in the cheese strains, and two different karyotypic profiles resulting from chromosomal rearrangements were observed. Together, these findings are consistent with clonal propagation of the cheese strains. Phenotypic trait variations were observed within the cheese population under stress conditions whereas the cider strain was found to have a much greater capacity for growth in all conditions tested.

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R2 non-long terminal repeat (non-LTR) retrotransposons are among the most extensively distributed mobile genetic elements in multicellular eukaryotes and show promise for applications in transgene supplementation of the human genome. They insert new gene copies into a conserved site in 28S ribosomal DNA with exquisite specificity. R2 clades are defined by the number of zinc fingers (ZFs) at the N terminus of the retrotransposon-encoded protein, postulated to additively confer DNA site specificity. Here, we illuminate general principles of DNA recognition by R2 N-terminal domains across and between clades, with extensive, specific recognition requiring only one or two compact domains. DNA-binding and protection assays demonstrate broadly shared as well as clade-specific DNA interactions. Gene insertion assays in cells identify the N-terminal domains sufficient for target-site insertion and reveal roles in second-strand cleavage or synthesis for clade-specific ZFs. Our results have implications for understanding evolutionary diversification of non-LTR retrotransposon insertion mechanisms and the design of retrotransposon-based gene therapies.

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By contrast to their conserved mammalian counterparts, plant long interspersed nuclear elements (LINEs) are highly variable, splitting into many low-copy families. Curiously, LINE families from the retrotransposable element (RTE) clade retain a stronger sequence conservation and hence reach higher copy numbers. The cause of this RTE-typical property is not yet understood, but would help clarify why some transposable elements are removed quickly, whereas others persist in plant genomes. Here, we bring forward a detailed study of RTE LINE structure, diversity and evolution in plants. For this, we argue that the nightshade family is the ideal taxon to follow the evolutionary trajectories of RTE LINEs, given their high abundance, recent activity and partnership to non-autonomous elements. Using bioinformatic, cytogenetic and molecular approaches, we detect 4029 full-length RTE LINEs across the Solanaceae. We finely characterize and manually curate a core group of 458 full-length LINEs in allotetraploid tobacco, show an integration event after polyploidization and trace hybridization by RTE LINE composition of parental genomes. Finally, we reveal the role of the untranslated regions (UTRs) as causes for the unique RTE LINE amplification and evolution pattern in plants. On the one hand, we detected a highly conserved motif at the 3' UTR, suggesting strong selective constraints acting on the RTE terminus. On the other hand, we observed successive rounds of 5' UTR cycling, constantly rejuvenating the promoter sequences. This interplay between exchangeable promoters and conserved LINE bodies and 3' UTR likely allows RTE LINEs to persist and thrive in plant genomes.

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BACKGROUND: Trypanosomatid genomes are colonized by active and inactive mobile DNA elements, such as LINE, SINE-like, SIDER and DIRE retrotransposons. These elements all share a 77-nucleotide-long sequence at their 5' ends, known as Pr77, which activates transcription, thereby generating abundant unspliced and translatable transcripts. However, transcription factors that mediates this process have still not been reported. METHODS: TATA-binding protein (TBP) and small nuclear RNA-activating protein 50 kDa (SNAP50) recombinant proteins and specific antibodies raised against them were generated. Protein capture assay, electrophoretic mobility-shift assays (EMSA) and EMSA competition assays carried out using these proteins and nuclear proteins of the parasite together to specific DNA sequences used as probes allowed detecting direct interaction of these transcription factors to Pr77 sequence. RESULTS: This study identified TBP and SNAP50 as part of the DNA-protein complex formed by the Pr77 promoter sequence and nuclear proteins of Trypanosoma cruzi. TBP establishes direct and specific contact with the Pr77 sequence, where the DPE and DPE downstream regions are docking sites with preferential binding. TBP binds cooperatively (Hill coefficient = 1.67) to Pr77 and to both strands of the Pr77 sequence, while the conformation of this highly structured sequence is not involved in TBP binding. Direct binding of SNAP50 to the Pr77 sequence is weak and may be mediated by protein-protein interactions through other trypanosomatid nuclear proteins. CONCLUSIONS: Identification of the transcription factors that mediate Pr77 transcription may help to elucidate how these retrotransposons are mobilized within the trypanosomatid genomes and their roles in gene regulation processes in this human parasite.

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Mobile elements are major regulators of genome evolution through their effects on genome size and chromosome structure in higher organisms. Non-long terminal repeat (non-LTR) retrotransposons, one of the subclasses of transposons, are specifically inserted into repetitive DNA sequences. While studies on the insertion of non-LTR retrotransposons into ribosomal RNA genes and other repetitive DNA sequences have been reported in the animal kingdom, studies in the plant kingdom are limited. Here, using FISH, we confirmed that Menolird18, a member of LINE (long interspersed nuclear element) in non-LTR retrotransposons and found in Cucumis melo, was inserted into ITS and ETS (internal and external transcribed spacers) regions of 18S rDNA in melon and cucumber. Beside the 18S rDNA regions, Menolird18 was also detected in all centromeric regions of melon, while it was located at pericentromeric and sub-telomeric regions in cucumber. The fact that FISH signals of Menolird18 were found in centromeric and rDNA regions of mitotic chromosomes suggests that Menolird18 is a rDNA and centromere-specific non-LTR retrotransposon in melon. Our findings are the first report on a non-LTR retrotransposon that is highly conserved in 2 different plant species, melon and cucumber. The clear distinction of chromosomal localization of Menolird18 in melon and cucumber implies that it might have been involved in the evolutionary processes of the melon (C. melo) and cucumber (C. sativus) genomes.

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Long interspersed elements (LINEs) replicate by target primed reverse transcription (TPRT). Insertion involves two half reactions. Each half reaction involves DNA cleavage followed by DNA synthesis. The linker region, located just beyond the reverse transcriptase in the LINE open reading frame, contains a set of predicted helices that may form an α-finger, followed by a gag-like zinc-knuckle. Point mutations of moderately conserved amino-acid residues in the presumptive α-finger severely impair the DNA endonuclease and reverse transcriptase activities of the integration reaction during both half reactions. Mutations in the gag-like zinc-knuckle also impair DNA cleavage and DNA synthesis in some instances. Mutations in core residues that presumably disrupt the protein structure of the presumptive α-finger and the gag-like zinc-knuckle lead to a promiscuous DNA endonuclease and protein-nucleic acid complexes that get stuck in the well during analysis. The linker region appears to function as a protein, DNA, and RNA conformational switching area. The linker is used to properly position nucleic acid substrates into the active sites of the reverse transcriptase and of the DNA endonuclease.

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Short interspersed nuclear elements (SINEs) are small, non-autonomous and heterogeneous retrotransposons that are widespread in plants. To explore the amplification dynamics and evolutionary history of SINE populations in representative deciduous tree species, we analyzed the genomes of the six following Salicaceae species: Populus deltoides, Populus euphratica, Populus tremula, Populus tremuloides, Populus trichocarpa, and Salix purpurea. We identified 11 Salicaceae SINE families (SaliS-I to SaliS-XI), comprising 27 077 full-length copies. Most of these families harbor segmental similarities, providing evidence for SINE emergence by reshuffling or heterodimerization. We observed two SINE groups, differing in phylogenetic distribution pattern, similarity and 3' end structure. These groups probably emerged during the 'salicoid duplication' (~65 million years ago) in the Salix-Populus progenitor and during the separation of the genus Salix (45-65 million years ago), respectively. In contrast to conserved 5' start motifs across species and SINE families, the 3' ends are highly variable in sequence and length. This extraordinary 3'-end variability results from mutations in the poly(A) tail, which were fixed by subsequent amplificational bursts. We show that the dissemination of newly evolved 3' ends is accomplished by a displacement of older motifs, leading to various 3'-end subpopulations within the SaliS families.

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The flavonoid compound anthocyanin is an important plant metabolite with nutritional and aesthetic value as well as anti-oxidative capacity. MYB transcription factors are key regulators of anthocyanin biosynthesis in plants. In pepper (Capsicum annuum), the CaAn2 gene, encoding an R2R3 MYB transcription factor, regulates anthocyanin biosynthesis. However, no functional study or structural analysis of functional and dysfunctional CaAn2 alleles has been performed. Here, to elucidate the function of CaAn2, we generated transgenic Nicotiana benthamiana and Arabidopsis thaliana plants expressing CaAn2. All of the tissues in these plants were purple. Promoter analysis of CaAn2 in purple C. annuum 'KC00134' plants revealed the insertion of a non-long terminal repeat (LTR) retrotransposon designated Ca-nLTR-A. To determine the promoter activity and functional domain of Ca-nLTR-A, various constructs carrying different domains of Ca-nLTR-A fused with GUS were transformed into N. benthamiana. Promoter analysis showed that the 3' untranslated region (UTR) of the second open reading frame of Ca-nLTR-A is responsible for CaAn2 expression in 'KC00134'. Sequence analysis of Ca-nLTR-A identified transcription factor binding sites known to regulate anthocyanin biosynthesis. This study indicates that insertion of a non-LTR retrotransposon in the promoter may activate expression of CaAn2 by recruiting transcription factors at the 3' UTR and thus provides the first example of exaptation of a non-LTR retrotransposon into a new promoter in plants.

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During the establishment of the duck EB66® cell line as a new cell substrate for vaccine production in industry, a very low level of reverse transcriptase (RT) activity was detected in the culture supernatant by product-enhanced RT assay but a whole battery of tests failed to evidence infectious particles. Results from extensive biochemical and physical testing demonstrated that RT activity was associated to an intracellular, non-enveloped and dense structure different from an infectious retroviruses. In silico analysis of Anas platyrhynchos genome revealed that the most likely candidates for encoding a ribonucleoprotein (RNP)-associated RT were nine copies of chicken repeat 1 (CR1)-like elements, belonging to the non-long terminal repeat retrotransposons. The presence of the full length Anas platyrhynchos chicken repeat 1-like sequence (APCR1) was confirmed in EB66® cells and the related ribonucleic acid was present in the RT-containing fraction of EB66® cells.

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BACKGROUND: Although most of long interspersed elements (LINEs), one class of non-LTR-retrotransposons, are integrated into the host genome randomely, some elements are retrotransposed into the specific sequences of the genomic regions, such as rRNA gene (rDNA) clusters, telomeric repeats and other repetitive sequenes. Most of the sequence-specific LINEs have been reported mainly among invertebrate species and shown to retrotranspose into the specific sequences in vivo and in vitro systems. Recenlty, 28S rDNA-specific LINE R2 elements are shown to be distributed among widespread vertebrate species, but the sequence-specific retrotransposition of R2 has never been demonstrated in vertebrates. RESULTS: Here we cloned a full length unit of R2 from medaka fish Oryzias latipes, named R2Ol, and engineered it to a targeted gene integration tool in zebrafish. By injecting R2Ol-encoding mRNA into zebrafish embryos, R2Ol retrotransposed precisely into the target site at high efficiency (98%) and was transmitted to the next generation at high frequency (50%). We also generated transgenic zebrafish carrying the enhanced green fluorescent protein (EGFP) reporter gene in 28S rDNA target by the R2Ol retrotransposition system. CONCLUSIONS: Sequence-specific LINE retrotransposes into the precise sequence using target primed reverse transcription (TPRT), possibly providing an alternative and effective targeted gene knockin method in vertebrates.

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INTRODUCTION: An important portion of the Trypanosoma cruzi genome is composed of mobile genetic elements, which are interspersed with genes on all chromosomes. The L1Tc non-LTR retrotransposon and its truncated version NARTc are the most highly represented and best studied of these elements. L1Tc is actively transcribed in all three forms of the Trypanosoma parasite and encodes the proteins that enable it to autonomously mobilize. This mini review discusses the enzymatic properties of L1Tc that enable its mobilization and possibly the mobilization of other non-autonomous retrotransposons in Trypanosoma. We also briefly review the Hepatitis Delta Virus-like autocatalytic and 2A self-cleaving viral-like sequences contained in L1Tc that regulate post-transcriptional properties such as relative protein abundance and mRNA stability. Special emphasis is placed on the Pr77 dual system, which is based on the RNA pol II-dependent internal promoter of L1Tc and NARTc and the HDV-like ribozyme activity encoded by the first 77 nucleotides of the element's DNA and RNA. The high degree of conservation of the Pr77 sequence, referred to as the "Pr77-hallmark", among different trypanosomatid retroelements suggests that these mobile elements are responsible for the distribution of regulatory sequences within the genome they inhabit. CONCLUSION: We also discuss how the involvement of L1Tc and NARTc in the gene regulatory processes of these parasites could justify their domestication and long-term coexistence in these ancient organisms.

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Even though lateral movements of transposons across families and even phyla within multicellular eukaryotic kingdoms have been found, little is known about transposon transfer between the kingdoms Animalia and Plantae. We discovered a novel non-LTR retrotransposon, AdLINE3, in a wild peanut species. Sequence comparisons and phylogenetic analyses indicated that AdLINE3 is a member of the RTE clade, originally identified in a nematode and rarely reported in plants. We identified RTE elements in 82 plants, spanning angiosperms to algae, including recently active elements in some flowering plants. RTE elements in flowering plants were likely derived from a single family we refer to as An-RTE. Interestingly, An-RTEs show significant DNA sequence identity with non-LTR retroelements from 42 animals belonging to four phyla. Moreover, the sequence identity of RTEs between two arthropods and two plants was higher than that of homologous genes. Phylogenetic and evolutionary analyses of RTEs from both animals and plants suggest that the An-RTE family was likely transferred horizontally into angiosperms from an ancient aphid(s) or ancestral arthropod(s). Notably, some An-RTEs were recruited as coding sequences of functional genes participating in metabolic or other biochemical processes in plants. This is the first potential example of horizontal transfer of transposons between animals and flowering plants. Our findings help to understand exchanges of genetic material between the kingdom Animalia and Plantae and suggest arthropods likely impacted on plant genome evolution.

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BACKGROUND: R2 elements are a clade of early branching Long Interspersed Elements (LINEs). LINEs are retrotransposable elements whose replication can have profound effects on the genomes in which they reside. No crystal or EM structures exist for the reverse transcriptase (RT) and linker regions of LINEs. RESULTS: Using limited proteolysis as a probe for globular domain structure, we show that the protein encoded by the Bombyx mori R2 element has two major globular domains: (1) a small globular domain consisting of the N-terminal zinc finger and Myb motifs, and (2) a large globular domain consisting of the RT, linker, and type II restriction-like endonuclease (RLE). Further digestion of the large globular domain occurred within the RT. Mapping these RT cleavages onto an updated model of the R2Bm RT indicated that the thumb of the RT was largely protected from proteolytic cleavage. The crystal structure of the large globular domain of Prp8, a eukaryotic splicing factor, was a major template used in building the R2Bm RT model, particularly the thumb region. The large fragment of Prp8 consists not only of a RT similar to R2Bm, but also an RLE and a linker connecting the two regions. The linker sequences adjacent to the RLE in LINEs and Prp8 share a set of two important α-helices and a (presumptive) knuckle/ßßα structural motif that are closely associated with the thumb. The RLEs of LINEs and Prp8 share a unique catalytic core residue spacing as well as other key residues. CONCLUSIONS: The protein encoded by RLE LINEs consists of two major globular domains. The larger of the two globular domain contains the RT, linker, and RLE and is similar to the large fragment of the spliceosomal protein Prp8. The similarities are suggestive of possible common ancestry.

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Short interspersed nuclear elements (SINEs) are non-autonomous transposable elements which are propagated by retrotransposition and constitute an inherent part of the genome of most eukaryotic species. Knowledge of heterogeneous and highly abundant SINEs is crucial for de novo (or improvement of) annotation of whole genome sequences. We scanned Poaceae genome sequences of six important cereals (Oryza sativa, Triticum aestivum, Hordeum vulgare, Panicum virgatum, Sorghum bicolor, Zea mays) and Brachypodium distachyon to examine the diversity and evolution of SINE populations. We comparatively analyzed the structural features, distribution, evolutionary relation and abundance of 32 SINE families and subfamilies within grasses, comprising 11 052 individual copies. The investigation of activity profiles within the Poaceae provides insights into their species-specific diversification and amplification. We found that Poaceae SINEs (PoaS) fall into two length categories: simple SINEs of up to 180 bp and dimeric SINEs larger than 240 bp. Detailed analysis at the nucleotide level revealed that multimerization of related and unrelated SINE copies is an important evolutionary mechanism of SINE formation. We conclude that PoaS families diversify by massive reshuffling between SINE families, likely caused by insertion of truncated copies, and provide a model for this evolutionary scenario. Twenty-eight of 32 PoaS families and subfamilies show significant conservation, in particular either in the 5' or 3' regions, across Poaceae species and share large sequence stretches with one or more other PoaS families.

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Non LTR retrotransposons (EhLINEs and EhSINEs) occupy 11% of the Entamoeba histolytica genome. Since promoter DNA methylation at cytosines has been correlated with transcriptional silencing of transposable elements in model organisms we checked whether this was the case in EhLINE1. We located promoter activity in a 841bp fragment at 5'-end of this element by luciferase reporter assay. From RNAseq and RT-PCR analyses we selected a transcriptionally active and silent copy to study cytosine DNA methylation of the promoter region by bisulfite sequencing. None of the cytosines were methylated in either copy. Further, we looked at methylation status of a few selected cytosines in all 5'-intact EhLINE1 copies by single nucleotide incorporation opposite cytosine in bisulfite-treated DNA, where dGTP would be incorporated if the cytosine was methylated. Again we did not find evidence of cytosine methylation, indicating that expression status of this element was not correlated with promoter DNA methylation. To test for any role of cytosine methylation in transcriptional regulation of the E. histolytica Hsp70 gene in which the promoter is fully methylated under normal growth conditions, we checked methylation status and found that the promoter remained fully methylated during heat-shock as well, although transcription was greatly enhanced by heat-shock, showing that cytosine methylation is not a repressive mark for EhHsp70. Our data present direct evidence that promoter methylation, a common mode of transposon silencing, is unlikely to be involved in transcriptional regulation of EhLINE1, and reinforce the conclusion that promoter DNA methylation may not be a major contributor to transcriptional regulation in E. histolytica.

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L1-seq is a high-throughput sequencing technique which is utilized to identify novel L1 insertions in genomic DNA samples of interest. Using special diagnostic nucleotides unique to the youngest and most active L1 sequence, we can amplify new somatic insertions. This technique has helped to establish the number of L1 insertions present in the general population as well as the variation among individuals with regard to their complement of active L1 elements. More recently, this technique has been employed to assess the level of retrotransposition occurring in various diseases such as cancer. These efforts try to establish a connection between the process of retrotransposition and disease development and/or progression.

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With the advent of new generations of high-throughput sequencing technologies, the catalog of human genome variants created by retrotransposon activity is expanding rapidly. However, despite these advances in describing L1 diversity and the fact that L1 must retrotranspose in the germline or prior to germline partitioning to be evolutionarily successful, direct assessment of de novo L1 retrotransposition in the germline or early embryogenesis has not been achieved for endogenous L1 elements. A direct study of de novo L1 retrotransposition into susceptible loci within sperm DNA (Freeman et al., Hum Mutat 32(8):978-988, 2011) suggested that the rate of L1 retrotransposition in the germline is much lower than previously estimated (<1 in 400 individuals versus 1 in 9 individuals (Kazazian, Nat Genet 22(2):130, 1999). Based on these revised estimates of the L1 retrotransposition rate, we modified the ATLAS L1 display technique (Badge et al., Am J Hum Genet 72(4):823-838, 2003) to investigate de novo L1 retrotransposition in human genomes. In this chapter, we describe how we combined a high-coverage ATLAS variant with high-throughput sequencing, achieving 11-25× sequence depth per single amplicon, to study L1 retrotransposition in whole genome amplified (WGA) DNAs.

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In vitro reverse transcriptase assays have been developed to monitor the presence and activity of ORF2p, an essential protein product of the LINE-1 retrotransposon (L1), in cellular fractions. We describe methods for expression and isolation of L1 ribonucleoprotein particles, and identification of ORF2p reverse transcriptase activity. Two independent methods are described: L1 element amplification protocol (LEAP) and direct L1 extension assay (DLEA). The first method involves cDNA synthesis by primer extension using dNTPs followed by a step of PCR amplification. The second method involves primer extension by incorporation of radiolabeled dTMPs followed by dot-blot or gel separation detection. Finally, we discuss the output and benefits of the two methods.

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Long interspersed elements (LINEs), or non-long-terminal repeat (LTR) retrotransposons, are mobile genetic elements that exist in the genomic DNA of most eukaryotes, comprising a considerable portion of the host chromosomes. LINEs constitute endogenous mutagens that cause insertional mutations in host chromosomes and have a large impact on host genome evolution. Despite their importance, however, the molecular mechanism of LINE retrotransposition is not fully understood. Several studies suggest that host proteins that participate in the repair of DNA breaks modulate LINE retrotransposition. Recently, we provided evidence that there are 2 distinct pathways-annealing and direct-that join the 5'-end of LINEs to host chromosomal DNA. These pathways appear to be used distinctively by zebrafish LINEs and the human L1 in DT40 cells. In HeLa cells, only the annealing pathway appears to be used. This implies that different characteristics of the 2 LINEs and also host factors dictate which pathway is selected. Here, we discuss the 5'-end-joining pathways of LINE retrotransposition and propose that the pathways of LINE integration adopt certain host repair factors.