To Be Mobile or Not: The Variety of Reverse Transcriptases and Their Recruitment by Host Genomes.

Reverse transcriptases (RT), or RNA-dependent DNA polymerases, are unorthodox enzymes that originally added a new angle to the conventional view of the unidirectional flow of genetic information in the cell from DNA to RNA to protein. First discovered in vertebrate retroviruses, RTs were since re-discovered in most eukaryotes, bacteria, and archaea, spanning essentially all domains of life. For retroviruses, RTs provide the ability to copy the RNA genome into DNA for subsequent incorporation into the host genome, which is essential for their replication and survival. In cellular organisms, most RT sequences originate from retrotransposons, the type of self-replicating genetic elements that rely on reverse transcription to copy and paste their sequences into new genomic locations. Some retroelements, however, can undergo domestication, eventually becoming a valuable addition to the overall repertoire of cellular enzymes. They can be beneficial yet accessory, like the diversity-generating elements, or even essential, like the telomerase reverse transcriptases. Nowadays, ever-increasing numbers of domesticated RT-carrying genetic elements are being discovered. It may be argued that domesticated RTs and reverse transcription in general is more widespread in cellular organisms than previously thought, and that many important cellular functions, such as chromosome end maintenance, may evolve from an originally selfish process of converting RNA into DNA.

Meeting report: transposable elements at the crossroads of evolution, health and disease 2023.

The conference "Transposable Elements at the Crossroads of Evolution, Health and Disease" was hosted by Keystone Symposia in Whistler, British Columbia, Canada, on September 3-6, 2023, and was organized by Kathleen Burns, Harmit Malik and Irina Arkhipova. The central theme of the meeting was the incredible diversity of ways in which transposable elements (TEs) interact with the host, from disrupting the existing genes and pathways to creating novel gene products and expression patterns, enhancing the repertoire of host functions, and ultimately driving host evolution. The meeting was organized into six plenary sessions and two afternoon workshops with a total of 50 invited and contributed talks, two poster sessions, and a career roundtable. The topics ranged from TE roles in normal and pathological processes to restricting and harnessing TE activity based on mechanistic insights gained from genetic, structural, and biochemical studies.

Shaping eukaryotic epigenetic systems by horizontal gene transfer.

DNA methylation constitutes one of the pillars of epigenetics, relying on covalent bonds for addition and/or removal of chemically distinct marks within the major groove of the double helix. DNA methyltransferases, enzymes which introduce methyl marks, initially evolved in prokaryotes as components of restriction-modification systems protecting host genomes from bacteriophages and other invading foreign DNA. In early eukaryotic evolution, DNA methyltransferases were horizontally transferred from bacteria into eukaryotes several times and independently co-opted into epigenetic regulatory systems, primarily via establishing connections with the chromatin environment. While C5-methylcytosine is the cornerstone of plant and animal epigenetics and has been investigated in much detail, the epigenetic role of other methylated bases is less clear. The recent addition of N4-methylcytosine of bacterial origin as a metazoan DNA modification highlights the prerequisites for foreign gene co-option into the host regulatory networks, and challenges the existing paradigms concerning the origin and evolution of eukaryotic regulatory systems.

An Overview of Best Practices for Transposable Element Identification, Classification, and Annotation in Eukaryotic Genomes.

Transposable elements (TEs) exert an increasingly diverse spectrum of influences on eukaryotic genome structure, function, and evolution. A deluge of genomic, transcriptomic, and proteomic data provides the foundation for turning essentially any non-model eukaryotic species into an emerging model to study any and all aspects of organismal biology, ultimately shaping future directions for biomedical, environmental, and biodiversity research. However, identification and annotation of the mobile genome component still lags behind the standards accepted for host gene annotation. To achieve the objective of providing every genome project with a comprehensive description of its mobilome component in addition to the standard genic and transcriptomic datasets, each step of TE identification, classification, and annotation should be focused on improving TE boundary designation, reducing identification error rates, and providing accurate information on the type and integrity of TE insertions. Here, we offer practical advice for generating TE models in de novo assemblies for non-model organisms, provide step-by-step instructions to guide inexperienced TE annotators through some of the commonly utilized TE analysis pipelines, and entertain suggestions for tool improvement which could be implemented by interested developers.

Bacterial N4-methylcytosine as an epigenetic mark in eukaryotic DNA.

DNA modifications are used to regulate gene expression and defend against invading genetic elements. In eukaryotes, modifications predominantly involve C5-methylcytosine (5mC) and occasionally N6-methyladenine (6mA), while bacteria frequently use N4-methylcytosine (4mC) in addition to 5mC and 6mA. Here we report that 4mC can serve as an epigenetic mark in eukaryotes. Bdelloid rotifers, tiny freshwater invertebrates with transposon-poor genomes rich in foreign genes, lack canonical eukaryotic C5-methyltransferases for 5mC addition, but encode an amino-methyltransferase, N4CMT, captured from bacteria >60 Mya. N4CMT deposits 4mC at active transposons and certain tandem repeats, and fusion to a chromodomain shapes its "histone-read-DNA-write" architecture recognizing silent chromatin marks. Furthermore, amplification of SETDB1 H3K9me3 histone methyltransferases yields variants preferentially binding 4mC-DNA, suggesting "DNA-read-histone-write" partnership to maintain chromatin-based silencing. Our results show how non-native DNA methyl groups can reshape epigenetic systems to silence transposons and demonstrate the potential of horizontal gene transfer to drive regulatory innovation in eukaryotes.

An Ancient Clade of Penelope-Like Retroelements with Permuted Domains Is Present in the Green Lineage and Protists, and Dominates Many Invertebrate Genomes.

Penelope-like elements (PLEs) are an enigmatic clade of retrotransposons whose reverse transcriptases (RTs) share a most recent common ancestor with telomerase RTs. The single ORF of canonical endonuclease (EN)+ PLEs encodes RT and a C-terminal GIY-YIG EN that enables intrachromosomal integration, whereas EN- PLEs lack EN and are generally restricted to chromosome termini. EN+ PLEs have only been found in animals, except for one case of horizontal transfer to conifers, whereas EN- PLEs occur in several kingdoms. Here, we report a new, deep-branching PLE clade with a permuted domain order, whereby an N-terminal GIY-YIG EN is linked to a C-terminal RT by a short domain with a characteristic CxC motif. These N-terminal EN+ PLEs share a structural organization, including pseudo-LTRs and complex tandem/inverted insertions, with canonical EN+ PLEs from Penelope/Poseidon, Neptune, and Nematis clades, and show insertion bias for microsatellites, but lack canonical hammerhead ribozyme motifs. However, their phylogenetic distribution is much broader. The Naiads, found in numerous invertebrate phyla, can reach tens of thousands of copies per genome. In spiders and clams, Naiads independently evolved to encode selenoproteins containing multiple selenocysteines. Chlamys, which lack the CCHH motif universal to PLE ENs, occur in green algae, spike mosses (targeting ribosomal DNA), and slime molds. Unlike canonical PLEs, RTs of N-terminal EN+ PLEs contain the insertion-in-fingers domain (IFD), strengthening the link between PLEs and telomerases. Additionally, we describe Hydra, a novel metazoan C-terminal EN+ clade. Overall, we conclude that PLE diversity, taxonomic distribution, and abundance are comparable with non-LTR and LTR-retrotransposons.

A Survey of Transposon Landscapes in the Putative Ancient Asexual Ostracod Darwinula stevensoni.

How asexual reproduction shapes transposable element (TE) content and diversity in eukaryotic genomes remains debated. We performed an initial survey of TE load and diversity in the putative ancient asexual ostracod Darwinula stevensoni. We examined long contiguous stretches of DNA in clones from a genomic fosmid library, totaling about 2.5 Mb, and supplemented these data with results on TE abundance and diversity from an Illumina draft genome. In contrast to other TE studies in putatively ancient asexuals, which revealed relatively low TE content, we found that at least 19% of the fosmid dataset and 26% of the genome assembly corresponded to known transposons. We observed a high diversity of transposon families, including LINE, gypsy, PLE, mariner/Tc, hAT, CMC, Sola2, Ginger, Merlin, Harbinger, MITEs and helitrons, with the prevalence of DNA transposons. The predominantly low levels of sequence diversity indicate that many TEs are or have recently been active. In the fosmid data, no correlation was found between telomeric repeats and non-LTR retrotransposons, which are present near telomeres in other taxa. Most TEs in the fosmid data were located outside of introns and almost none were found in exons. We also report an N-terminal Myb/SANT-like DNA-binding domain in site-specific R4/Dong non-LTR retrotransposons. Although initial results on transposable loads need to be verified with high quality draft genomes, this study provides important first insights into TE dynamics in putative ancient asexual ostracods.

Evolutionary dynamics of transposable elements in bdelloid rotifers.

Transposable elements (TEs) are selfish genomic parasites whose ability to spread autonomously is facilitated by sexual reproduction in their hosts. If hosts become obligately asexual, TE frequencies and dynamics are predicted to change dramatically, but the long-term outcome is unclear. Here, we test current theory using whole-genome sequence data from eight species of bdelloid rotifers, a class of invertebrates in which males are thus far unknown. Contrary to expectations, we find a variety of active TEs in bdelloid genomes, at an overall frequency within the range seen in sexual species. We find no evidence that TEs are spread by cryptic recombination or restrained by unusual DNA repair mechanisms. Instead, we find that that TE content evolves relatively slowly in bdelloids and that gene families involved in RNAi-mediated TE suppression have undergone significant expansion, which might mitigate the deleterious effects of active TEs and compensate for the consequences of long-term asexuality.

Genomic signatures of recombination in a natural population of the bdelloid rotifer Adineta vaga.

Sexual reproduction is almost ubiquitous among extant eukaryotes. As most asexual lineages are short-lived, abandoning sex is commonly regarded as an evolutionary dead end. Still, putative anciently asexual lineages challenge this view. One of the most striking examples are bdelloid rotifers, microscopic freshwater invertebrates believed to have completely abandoned sexual reproduction tens of Myr ago. Here, we compare whole genomes of 11 wild-caught individuals of the bdelloid rotifer Adineta vaga and present evidence that some patterns in its genetic variation are incompatible with strict clonality and lack of genetic exchange. These patterns include genotype proportions close to Hardy-Weinberg expectations within loci, lack of linkage disequilibrium between distant loci, incongruent haplotype phylogenies across the genome, and evidence for hybridization between divergent lineages. Analysis of triallelic sites independently corroborates these findings. Our results provide evidence for interindividual genetic exchange and recombination in A. vaga, a species previously thought to be anciently asexual.

Metagenome Proteins and Database Contamination.

Continued influx of metagenome-derived proteins with misannotated taxonomy into conventional databases, including RefSeq, threatens to eliminate the value of taxonomy identifiers. To prevent this, urgent efforts should be undertaken by submitters of metagenomic data sets as well as by database managers.

Adaptation of gene loci to heterochromatin in the course of Drosophila evolution is associated with insulator proteins.

Pericentromeric heterochromatin is generally composed of repetitive DNA forming a transcriptionally repressive environment. Dozens of genes were embedded into pericentromeric heterochromatin during evolution of Drosophilidae lineage while retaining activity. However, factors that contribute to insusceptibility of gene loci to transcriptional silencing remain unknown. Here, we find that the promoter region of genes that can be embedded in both euchromatin and heterochromatin exhibits a conserved structure throughout the Drosophila phylogeny and carries motifs for binding of certain chromatin remodeling factors, including insulator proteins. Using ChIP-seq data, we demonstrate that evolutionary gene relocation between euchromatin and pericentric heterochromatin occurred with preservation of sites of insulation of BEAF-32 in evolutionarily distant species, i.e. D. melanogaster and D. virilis. Moreover, promoters of virtually all protein-coding genes located in heterochromatin in D. melanogaster are enriched with insulator proteins BEAF-32, GAF and dCTCF. Applying RNA-seq of a BEAF-32 mutant, we show that the impairment of BEAF-32 function has a complex effect on gene expression in D. melanogaster, affecting even those genes that lack BEAF-32 association in their promoters. We propose that conserved intrinsic properties of genes, such as sites of insulation near the promoter regions, may contribute to adaptation of genes to the heterochromatic environment and, hence, facilitate the evolutionary relocation of genes loci between euchromatin and heterochromatin.

A partial genome assembly of the miniature parasitoid wasp, Megaphragma amalphitanum.

Body size reduction, also known as miniaturization, is an important evolutionary process that affects a number of physiological and phenotypic traits and helps animals conquer new ecological niches. However, this process is poorly understood at the molecular level. Here, we report genomic and transcriptomic features of arguably the smallest known insect-the parasitoid wasp, Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae). In contrast to expectations, we find that the genome and transcriptome sizes of this parasitoid wasp are comparable to other members of the Chalcidoidea superfamily. Moreover, compared to other chalcid wasps the gene content of M. amalphitanum is remarkably conserved. Intriguingly, we observed significant changes in M. amalphitanum transposable element dynamics over time, in which an initial burst was followed by suppression of activity, possibly due to a recent reinforcement of the genome defense machinery. Overall, while the M. amalphitanum genomic data reveal certain features that may be linked to the unusual biological properties of this organism, miniaturization is not associated with a large decrease in genome complexity.

Giant Transposons in Eukaryotes: Is Bigger Better?

Transposable elements (TEs) are ubiquitous in both prokaryotes and eukaryotes, and the dynamic character of their interaction with host genomes brings about numerous evolutionary innovations and shapes genome structure and function in a multitude of ways. In traditional classification systems, TEs are often being depicted in simplistic ways, based primarily on the key enzymes required for transposition, such as transposases/recombinases and reverse transcriptases. Recent progress in whole-genome sequencing and long-read assembly, combined with expansion of the familiar range of model organisms, resulted in identification of unprecedentedly long transposable units spanning dozens or even hundreds of kilobases, initially in prokaryotic and more recently in eukaryotic systems. Here, we focus on such oversized eukaryotic TEs, including retrotransposons and DNA transposons, outline their complex and often combinatorial nature and closely intertwined relationship with viruses, and discuss their potential for participating in transfer of long stretches of DNA in eukaryotes.

Meeting report: mobile genetic elements and genome plasticity 2018.

The Mobile Genetic Elements and Genome Plasticity conference was hosted by Keystone Symposia in Santa Fe, NM USA, February 11-15, 2018. The organizers were Marlene Belfort, Evan Eichler, Henry Levin and Lynn Maquat. The goal of this conference was to bring together scientists from around the world to discuss the function of transposable elements and their impact on host species. Central themes of the meeting included recent innovations in genome analysis and the role of mobile DNA in disease and evolution. The conference included 200 scientists who participated in poster presentations, short talks selected from abstracts, and invited talks. A total of 58 talks were organized into eight sessions and two workshops. The topics varied from mechanisms of mobilization, to the structure of genomes and their defense strategies to protect against transposable elements.

Biochemical properties of bacterial reverse transcriptase-related (rvt) gene products: multimerization, protein priming, and nucleotide preference.

Cellular reverse transcriptase-related (rvt) genes represent a novel class of reverse transcriptases (RTs), which are only distantly related to RTs of retrotransposons and retroviruses, but, similarly to telomerase RTs, are immobilized in the genome as single-copy genes. They have been preserved by natural selection throughout the evolutionary history of large taxonomic groups, including most fungi, a few plants and invertebrates, and even certain bacteria, being the only RTs present across different domains of life. Bacterial rvt genes are exceptionally rare but phylogenetically related, consistent with common origin of bacterial rvt genes rather than eukaryote-to-bacteria transfer. To investigate biochemical properties of bacterial RVTs, we conducted in vitro studies of recombinant HaRVT protein from the filamentous gliding bacterium Herpetosiphon aurantiacus (Chloroflexi). Although HaRVT does not utilize externally added standard primer-template combinations, in the presence of divalent manganese, it can polymerize very short products, using dNTPs rather than NTPs, with a strong preference for dCTP incorporation. Furthermore, we investigated the highly conserved N- and C-terminal domains, which distinguish RVT proteins from other RTs. We show that the N-terminal coiled-coil motif, which is present in nearly all RVTs, is responsible for the ability of HaRVT to multimerize in solution, forming up to octamers. The C-terminal domain may be capable of protein priming, which is abolished by site-directed mutagenesis of the catalytic aspartate and greatly reduced in the absence of the conserved tyrosine residues near the C-terminus. The unusual biochemical properties displayed by RVT in vitro will provide the basis for understanding its biological function in vivo.

Transposable elements and polyploid evolution in animals.

Polyploidy in animals is much less common than in plants, where it is thought to be pervasive in all higher plant lineages. Recent studies have highlighted the impact of polyploidization and the associated process of diploidy restoration on the evolution and speciation of selected taxonomic groups in the animal kingdom: from vertebrates represented by salmonid fishes and African clawed frogs to invertebrates represented by parasitic root-knot nematodes and bdelloid rotifers. In this review, we focus on the unique and diverse roles that transposable elements may play in these processes, from marking and diversifying subgenome-specific chromosome sets before hybridization, to influencing genome restructuring during rediploidization, to affecting subgenome-specific regulatory evolution, and occasionally providing opportunities for domestication and gene amplification to restore and improve functionality. There is still much to be learned from the future comparative genomic studies of chromosome-sized and haplotype-aware assemblies, and from postgenomic studies elucidating genetic and epigenetic regulatory phenomena across short and long evolutionary distances in the metazoan tree of life.

Neutral Theory, Transposable Elements, and Eukaryotic Genome Evolution.

Among the multitude of papers published yearly in scientific journals, precious few publications may be worth looking back in half a century to appreciate the significance of the discoveries that would later become common knowledge and get a chance to shape a field or several adjacent fields. Here, Kimura's fundamental concept of neutral mutation-random drift, which was published 50 years ago, is re-examined in light of its pervasive influence on comparative genomics and, more specifically, on the contribution of transposable elements to eukaryotic genome evolution.

Using bioinformatic and phylogenetic approaches to classify transposable elements and understand their complex evolutionary histories.

In recent years, much attention has been paid to comparative genomic studies of transposable elements (TEs) and the ensuing problems of their identification, classification, and annotation. Different approaches and diverse automated pipelines are being used to catalogue and categorize mobile genetic elements in the ever-increasing number of prokaryotic and eukaryotic genomes, with little or no connectivity between different domains of life. Here, an overview of the current picture of TE classification and evolutionary relationships is presented, updating the diversity of TE types uncovered in sequenced genomes. A tripartite TE classification scheme is proposed to account for their replicative, integrative, and structural components, and the need to expand in vitro and in vivo studies of their structural and biological properties is emphasized. Bioinformatic studies have now become front and center of novel TE discovery, and experimental pursuits of these discoveries hold great promise for both basic and applied science.

Giant Reverse Transcriptase-Encoding Transposable Elements at Telomeres.

Transposable elements are omnipresent in eukaryotic genomes and have a profound impact on chromosome structure, function and evolution. Their structural and functional diversity is thought to be reasonably well-understood, especially in retroelements, which transpose via an RNA intermediate copied into cDNA by the element-encoded reverse transcriptase, and are characterized by a compact structure. Here, we report a novel type of expandable eukaryotic retroelements, which we call Terminons. These elements can attach to G-rich telomeric repeat overhangs at the chromosome ends, in a process apparently facilitated by complementary C-rich repeats at the 3'-end of the RNA template immediately adjacent to a hammerhead ribozyme motif. Terminon units, which can exceed 40 kb in length, display an unusually complex and diverse structure, and can form very long chains, with host genes often captured between units. As the principal polymerizing component, Terminons contain Athena reverse transcriptases previously described in bdelloid rotifers and belonging to the enigmatic group of Penelope-like elements, but can additionally accumulate multiple cooriented ORFs, including DEDDy 3'-exonucleases, GDSL esterases/lipases, GIY-YIG-like endonucleases, rolling-circle replication initiator (Rep) proteins, and putatively structural ORFs with coiled-coil motifs and transmembrane domains. The extraordinary length and complexity of Terminons and the high degree of interfamily variability in their ORF content challenge the current views on the structural organization of eukaryotic retroelements, and highlight their possible connections with the viral world and the implications for the elevated frequency of gene transfer.

LTR-Retrotransposons from Bdelloid Rotifers Capture Additional ORFs Shared between Highly Diverse Retroelement Types.

Rotifers of the class Bdelloidea, microscopic freshwater invertebrates, possess a highlydiversified repertoire of transposon families, which, however, occupy less than 4% of genomic DNA in the sequenced representative Adineta vaga. We performed a comprehensive analysis of A. vaga retroelements, and found that bdelloid long terminal repeat (LTR)retrotransposons, in addition to conserved open reading frame (ORF) 1 and ORF2 corresponding to gag and pol genes, code for an unusually high variety of ORF3 sequences. Retrovirus-like LTR families in A. vaga belong to four major lineages, three of which are rotiferspecific and encode a dUTPase domain. However only one lineage contains a canonical envlike fusion glycoprotein acquired from paramyxoviruses (non-segmented negative-strand RNA viruses), although smaller ORFs with transmembrane domains may perform similar roles. A different ORF3 type encodes a GDSL esterase/lipase, which was previously identified as ORF1 in several clades of non-LTR retrotransposons, and implicated in membrane targeting. Yet another ORF3 type appears in unrelated LTR-retrotransposon lineages, and displays strong homology to DEDDy-type exonucleases involved in 3'-end processing of RNA and single-stranded DNA. Unexpectedly, each of the enzymatic ORF3s is also associated with different subsets of Penelope-like Athena retroelement families. The unusual association of the same ORF types with retroelements from different classes reflects their modular structure with a high degree of flexibility, and points to gene sharing between different groups of retroelements.