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

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Current Genetics 64 (2018): 1287-1301, doi:10.1007/s00294-018-0844-6.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. Further, 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.This work was supported by the U.S. National Science Foundation grant MCB-1121334 to I.A.2019-05-1

Giant reverse transcriptase-encoding transposable elements at telomeres

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here by permission of Oxford University Press for personal use, not for redistribution. The definitive version was published in Molecular Biology and Evolution 34 (2017): 2245–2257, doi:10.1093/molbev/msx159.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 co-oriented 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 inter-family 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.This work was supported by the National Institutes of Health (grant GM111917 to I.A.).2018-05-3

Transposable elements and polyploid evolution in animals

© The Author(s), 2018. This is the author's version of the work and is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Current Opinion in Genetics & Development 49 (2018): 115-123, doi:10.1016/j.gde.2018.04.003.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 prior to 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 post-genomic studies elucidating genetic and epigenetic regulatory phenomena across short and long evolutionary distances in the metazoan tree of life.Work in the laboratory is supported by R01GM111917 from the U.S. National Institutes of Health to I.A.2019-04-3

Endonuclease-containing Penelope retrotransposons in the bdelloid rotifer Adineta vaga exhibit unusual structural features and play a role in expansion of host gene families

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Mobile DNA 4 (2013): 19, doi:10.1186/1759-8753-4-19.Penelope-like elements (PLEs) are an enigmatic group of retroelements sharing a common ancestor with telomerase reverse transcriptases. In our previous studies, we identified endonuclease-deficient PLEs that are associated with telomeres in bdelloid rotifers, small freshwater invertebrates best known for their long-term asexuality and high foreign DNA content. Completion of the high-quality draft genome sequence of the bdelloid rotifer Adineta vaga provides us with the opportunity to examine its genomic transposable element (TE) content, as well as TE impact on genome function and evolution. We performed an exhaustive search of the A. vaga genome assembly, aimed at identification of canonical PLEs combining both the reverse transcriptase (RT) and the GIY-YIG endonuclease (EN) domains. We find that the RT/EN-containing Penelope families co-exist in the A. vaga genome with the EN-deficient RT-containing Athena retroelements. Canonical PLEs are present at very low copy numbers, often as a single-copy, and there is no evidence that they might preferentially co-mobilize EN-deficient PLEs. We also find that Penelope elements can participate in expansion of A. vaga multigene families via trans-action of their enzymatic machinery, as evidenced by identification of intron-containing host genes framed by the Penelope terminal repeats and characteristic target-site duplications generated upon insertion. In addition, we find that Penelope open reading frames (ORFs) in several families have incorporated long stretches of coding sequence several hundred amino acids (aa) in length that are highly enriched in asparagine residues, a phenomenon not observed in other retrotransposons. Our results show that, despite their low abundance and low transcriptional activity in the A. vaga genome, endonuclease-containing Penelope elements can participate in expansion of host multigene families. We conclude that the terminal repeats represent the cis-acting sequences required for mobilization of the intervening region in trans by the Penelope-encoded enzymatic activities. We also hypothesize that the unusual capture of long N-rich segments by the Penelope ORF occurs as a consequence of peculiarities of its replication mechanism. These findings emphasize the unconventional nature of Penelope retrotransposons, which, in contrast to all other retrotransposon types, are capable of dispersing intron-containing genes, thereby questioning the validity of traditional estimates of gene retrocopies in PLE-containing eukaryotic genomes.This research was supported by grants MCB-0821956 and MCB-1121334 from the U.S. National Science Foundation to I.A

Rotifer rDNA-specific R9 retrotransposable elements generate an exceptionally long target site duplication upon insertion

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Gene 448 (2009): 145-150, doi:10.1016/j.gene.2009.08.016.Ribosomal DNA genes in many eukaryotes contain insertions of non-LTR retrotransposable elements belonging to the R2 clade. These elements persist in the host genomes by inserting site-specifically into multicopy target sites, thereby avoiding random disruption of single-copy host genes. Here we describe R9 retrotransposons from the R2 clade in the 28S RNA genes of bdelloid rotifers, small freshwater invertebrate animals best known for their long-term asexuality and for their ability to survive repeated cycles of desiccation and rehydration. While the structural organization of R9 elements is highly similar to that of other members of the R2 clade, they are characterized by two distinct features: sitespecific insertion into a previously unreported target sequence within the 28S gene, and an unusually long target site duplication of 126 bp. We discuss the implications of these findings in the context of bdelloid genome organization and the mechanisms of target-primed reverse transcription.This work was supported by the U.S. National Science Foundation grant MCB-0821956 to I.A

Massive Horizontal Gene Transfer in Bdelloid Rotifers

Horizontal gene transfer in metazoans has been documented in only a few species and is usually associated with endosymbiosis or parasitism. By contrast, in bdelloid rotifers we found many genes that appear to have originated in bacteria, fungi, and plants, concentrated in telomeric regions along with diverse mobile genetic elements. Bdelloid proximal gene-rich regions, however, appeared to lack foreign genes, thereby resembling those of model metazoan organisms. Some of the foreign genes were defective, whereas others were intact and transcribed; some of the latter contained functional spliceosomal introns. One such gene, apparently of bacterial origin, was overexpressed in Escherichia coli and yielded an active enzyme. The capture and functional assimilation of exogenous genes may represent an important force in bdelloid evolution.Molecular and Cellular Biolog

Mobile genetic elements : in silico, in vitro, in vivo

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in Molecular Ecology 25 (2016): 1027-1031, doi:10.1111/mec.13543.Mobile genetic elements (MGEs), also called transposable elements (TEs), represent universal components of most genomes and are intimately involved in nearly all aspects of genome organization, function, and evolution. However, there is currently a gap between fast-paced TE discovery in silico, stimulated by exponential growth of comparative genomic studies, and a limited number of experimental models amenable to more traditional in vitro and in vivo studies of structural, mechanistic, and regulatory properties of diverse MGEs. Experimental and computational scientists came together to bridge this gap at a recent conference, “Mobile Genetic Elements: in silico, in vitro, in vivo,” held at the Marine Biological Laboratory (MBL) in Woods Hole, MA, USA.Research in the authors’ laboratories is supported by NSF MCB-1121334 and NIH R01 GM111917 (I.A.), and by NIH R01 GM101989 and R21 AI117593 (P.R.)2016-11-3

A subtelomeric non-LTR retrotransposon Hebe in the bdelloid rotifer Adineta vaga is subject to inactivation by deletions but not 5' truncations

<p>Abstract</p> <p>Background</p> <p>Rotifers of the class Bdelloidea are microscopic freshwater invertebrates best known for: their capacity for anhydrobiosis; the lack of males and meiosis; and for the ability to capture genes from other non-metazoan species. Although genetic exchange between these animals might take place by non-canonical means, the overall lack of meiosis and syngamy should greatly impair the ability of transposable elements (TEs) to spread in bdelloid populations. Previous studies demonstrated that bdelloid chromosome ends, in contrast to gene-rich regions, harbour various kinds of TEs, including specialized telomere-associated retroelements, as well as DNA TEs and retrovirus-like retrotransposons which are prone to horizontal transmission. Vertically-transmitted retrotransposons have not previously been reported in bdelloids and their identification and studies of the patterns of their distribution and evolution could help in the understanding of the high degree of TE compartmentalization within bdelloid genomes.</p> <p>Results</p> <p>We identified and characterized a non-long terminal repeat (LTR) retrotransposon residing primarily in subtelomeric regions of the genome in the bdelloid rotifer <it>Adineta vaga</it>. Contrary to the currently prevailing views on the mode of proliferation of non-LTR retrotransposons, which results in frequent formation of 5'-truncated ('dead-on-arrival') copies due to the premature disengagement of the element-encoded reverse transcriptase from its template, this non-LTR element, <it>Hebe</it>, is represented only by non-5'-truncated copies. Most of these copies, however, were subject to internal deletions associated with microhomologies, a hallmark of non-homologous end-joining events.</p> <p>Conclusions</p> <p>The non-LTR retrotransposon <it>Hebe </it>from the bdelloid rotifer <it>A. vaga </it>was found to undergo frequent microhomology-associated deletions, rather than 5'-terminal truncations characteristic of this class of retrotransposons, and to exhibit preference for telomeric localization. These findings represent the first example of a vertically transmitted putatively deleterious TE in bdelloids, and may indicate the involvement of microhomology-mediated non-homologous end-joining in desiccation-induced double-strand break repair at the genome periphery.</p

LTR-retrotransposons from Bdelloid rotifers capture additional ORFs shared between highly diverse retroelement types

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Viruses 9 (2017): 78, doi:10.3390/v9040078.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.This work was supported by the National Institutes of Health grant GM111917 to I.A.; A.K. was supported by the Research Experiences for Undergraduates supplement to the National Science Foundation grant MCB-1121334 to I.A