Maintenance of multiple lineages of R1 and R2 retrotransposable elements in the ribosomal RNA gene loci of Nasonia.

Sequencing reads from the Nasonia genome project were used to study the ribosomal RNA gene loci and the retrotransposons R1 and R2 that insert specifically into the 28S genes. Five highly divergent R1 and five highly divergent R2 families were identified in the three sequenced species, as well as a non-autonomous element that appears to use the retrotransposition machinery of R1. A duplication of the R1 target site within the spacer region of the rDNA units was also found to be extensively utilized by R1 elements. We document numerous instances where the R1 and R2 families appropriated parts of the retrotransposition machinery of other lineages and speculate that this enables rapid adaptation and the maintenance of multiple R1 and R2 families.

Competition between R1 and R2 transposable elements in the 28S rRNA genes of insects.

R1 and R2 are non-LTR retrotransposons that insert in the 28S rRNA genes of arthropods. R1 elements insert into a site that is 74 bp downstream of the R2 insertion site, thus the presence of an R2 in the same 28S gene may inhibit the expression of R1. Consistent with such a suggestion, the R1 elements of Drosophila melanogaster have a strong bias against inserting into 28S genes already containing an R2 element. R2 elements, on the other hand, are only 2-3 fold inhibited from inserting into a 28S gene already containing an R1. D. melanogaster R1 elements are unusual in that they generate a 23-bp deletion of the target site upstream of the insertion. Using in vitro assays developed to study R2 integration, we show that the presence of R1 sequences 51 bp downstream of the R2 insertion site changes the nucleosomal structure that can be formed by the R2 target site. The R2 endonuclease is inhibited from cleaving these altered nucleosomes. We suggest that R1 elements have been selected to make this large deletion of the 28S gene to block the insertion of an upstream R2 element. These findings are consistent with the model that R1 and R2 are in competition for the limited number of insertion sites available within their host's genome.

Dynamics of R1 and R2 elements in the rDNA locus of Drosophila simulans.

The mobile elements R1 and R2 insert specifically into the rRNA gene locus (rDNA locus) of arthropods, a locus known to undergo concerted evolution, the recombinational processes that preserve the sequence homogeneity of all repeats. To monitor how rapidly individual R1 and R2 insertions are turned over in the rDNA locus by these processes, we have taken advantage of the many 5' truncation variants that are generated during the target-primed reverse transcription mechanism used by these non-LTR retrotransposons for their integration. A simple PCR assay was designed to reveal the pattern of the 5' variants present in the rDNA loci of individual X chromosomes in a population of Drosophila simulans. Each rDNA locus in this population was found to have a large, unique collection of 5' variants. Each variant was present at low copy number, usually one copy per chromosome, and was seldom distributed to other chromosomes in the population. The failure of these variants to spread to other units in the same rDNA locus suggests a strong recombinational bias against R1 and R2 that results in the individual copies of these elements being rapidly lost from the rDNA locus. This bias suggests a significantly higher frequency of R1 and R2 retrotransposition than we have previously suggested.

Phylogenetic analysis of ribonuclease H domains suggests a late, chimeric origin of LTR retrotransposable elements and retroviruses.

We have conducted a phylogenetic analysis of the Ribonuclease HI (RNH) domains present in Eubacteria, Eukarya, all long-term repeat (LTR)-bearing retrotransposons, and several late-branching clades of non-LTR retrotransposons. Analysis of this simple yet highly conserved enzymatic domain from these disparate sources provides surprising insights into the evolution of eukaryotic retrotransposons. First, it indicates that the lineage of elements leading to vertebrate retroviruses acquired a new RNH domain either from non-LTR retrotransposons or from a eukaryotic host genome. The preexisting retroviral RNH domain degenerated to become the tether (connection) domain of the reverse transcriptase (RT)-RNH complex. Second, it indicates that all LTR retrotransposons arose in eukaryotes well after the origin of the non-LTR retrotransposons. Because of the younger age of the LTR retrotransposons, their complex structure, and the absence of any prokaryotic precursors, we propose that the LTR retrotransposons originated as a fusion between a DNA-mediated transposon and a non-LTR retrotransposon. The resulting two-step mechanism of LTR retrotransposition, in which RNA is reverse transcribed away from the chromosomal target site, rather than directly onto the target site, was probably an adaptation to the uncoupling of transcription and translation in eukaryotic cells.

Multiple lineages of R1 retrotransposable elements can coexist in the rDNA loci of Drosophila.

R1 non-long terminal repeat retrotransposable elements insert specifically into the 28S rRNA genes of arthropods. One aspect of R1 evolution that has been difficult to explain is the presence of divergent lineages of R1 in the rDNA loci of the same species. Multiple lineages should compete for a limited number of insertion sites, in addition to being subject to the concerted evolution processes homogenizing the rRNA genes. The presence of multiple lineages suggests either the ability of the elements to overcome these factors and diverge within rDNA loci, or the introduction of new lineages by horizontal transmission. To address this issue, we attempted to characterize the complete set of R1 elements in the rDNA locus from five Drosophila species groups (melanogaster, obscura, testacea, quinaria, and repleta). Two major R1 lineages, A and B, that diverged about 100 MYA were found to exist in Drosophila. Elements of the A lineage were found in all 35 Drosophila species tested, while elements of the B lineage were found in only 11 species from three species groups. Phylogenetic analysis of the R1 elements, supported by comparison of their rates of nucleotide sequence substitution, revealed that both the A and the B lineages have been maintained by vertical descent. The B lineage was less stable and has undergone numerous, independent elimination events, while the A lineage has diverged into three sublineages, which were, in turn, differentially stable. We conclude that while the differential retention of multiple lineages greatly complicates its phylogenetic history, the available R1 data continue to be consistent with the strict vertical descent of these elements.

Ten-kilodalton domain in Ty3 Gag3-Pol3p between PR and RT is dispensable for Ty3 transposition.

Ty3 is a gypsy-type, retrovirus-like element found in the budding yeast Saccharomyces cerevisiae. In cells overexpressing Ty3 under the GAL1 upstream activation sequence, Ty3 RNA, proteins, and DNA are made. Elucidation of the molecular masses and amino-terminal sequences of protease and reverse transcriptase indicated the existence of an additional intervening domain, designated J, in the Ty3 Gag3-Pol3p polyprotein. A region analogous to J can be found in many retrotransposable elements closely related to Ty3; however, J does not correspond to any of the highly conserved retroviral protein domains. Ty3 mutants deleted for the J-coding region showed moderately reduced transposition frequency but greatly reduced levels of Ty3 DNA. These results show that under galactose regulation, the Ty3 J domain is not absolutely essential.

Poised for contagion: evolutionary origins of the infectious abilities of invertebrate retroviruses.

Phylogenetic analyses suggest that long-terminal repeat (LTR) bearing retrotransposable elements can acquire additional open-reading frames that can enable them to mediate infection. Whereas this process is best documented in the origin of the vertebrate retroviruses and their acquisition of an envelope (env) gene, similar independent events may have occurred in insects, nematodes, and plants. The origins of env-like genes are unclear, and are often masked by the antiquity of the original acquisitions and by their rapid rate of evolution. In this report, we present evidence that in three other possible transitions of LTR retrotransposons to retroviruses, an envelope-like gene was acquired from a viral source. First, the gypsy and related LTR retrotransposable elements (the insect errantiviruses) have acquired their envelope-like gene from a class of insect baculoviruses (double-stranded DNA viruses with no RNA stage). Second, the Cer retroviruses in the Caenorhabditis elegans genome acquired their envelope gene from a Phleboviral (single ambisense-stranded RNA viruses) source. Third, the Tas retroviral envelope (Ascaris lumricoides) may have been obtained from Herpesviridae (double-stranded DNA viruses, no RNA stage). These represent the only cases in which the env gene of a retrovirus has been traced back to its original source. This has implications for the evolutionary history of retroviruses as well as for the potential ability of all LTR-retrotransposable elements to become infectious agents.

Putative telomerase catalytic subunits from Giardia lamblia and Caenorhabditis elegans.

Eukaryotic chromosomes end in short nucleotide repeats that are added by the enzyme telomerase. The catalytic subunit of telomerase has been shown to be most closely related in sequence to reverse transcriptases encoded by eukaryotic retrotransposable elements. This raises the question as to whether the telomerase subunit was present in the first eukaryotes or was derived during early eukaryote evolution from the replication machinery of a retrotransposable element. We present the sequence of a putative telomerase catalytic subunit from the diplomonad parasite, Giardia lamblia. The G. lamblia subunit appears to have most of the characteristics of other sequenced telomerases, except that it lacks the conserved telomerase-specific 'T' motif previously identified in other eukaryotic genes. Searching genomic databases with the G. lamblia sequence, we also identified a potential telomerase catalytic subunit from Caenorhabditis elegans. The C. elegans subunit is uncharacteristically short, and lacks several motifs found in all other telomerases. The identification of a G. lamblia telomerase similar to that of most other eukaryotes suggests that telomerase dates back to the earliest extant marker of eukaryotic evolution. The atypical C. elegans telomerase, on the other hand, raises intriguing biochemical questions concerning sub-domains of the telomerase catalytic subunit previously considered indispensable. The enzymatic machinery for telomere formation in C. elegans is likely to differ substantially from that of other eukaryotes.

NeSL-1, an ancient lineage of site-specific non-LTR retrotransposons from Caenorhabditis elegans.

Phylogenetic analyses of non-LTR retrotransposons suggest that all elements can be divided into 11 lineages. The 3 oldest lineages show target site specificity for unique locations in the genome and encode an endonuclease with an active site similar to certain restriction enzymes. The more "modern" non-LTR lineages possess an apurinic endonuclease-like domain and generally lack site specificity. The genome sequence of Caenorhabditis elegans reveals the presence of a non-LTR retrotransposon that resembles the older elements, in that it contains a single open reading frame with a carboxyl-terminal restriction-like endonuclease domain. Located near the N-terminal end of the ORF is a cysteine protease domain not found in any other non-LTR element. The N2 strain of C. elegans appears to contain only one full-length and several 5' truncated copies of this element. The elements specifically insert in the Spliced leader-1 genes; hence the element has been named NeSL-1 (Nematode Spliced Leader-1). Phylogenetic analysis confirms that NeSL-1 branches very early in the non-LTR lineage and that it represents a 12th lineage of non-LTR elements. The target specificity of NeSL-1 for the spliced leader exons and the similarity of its structure to that of R2 elements leads to a simple model for its expression and retrotransposition.

Integration of Bombyx mori R2 sequences into the 28S ribosomal RNA genes of Drosophila melanogaster.

R2 non-long-terminal-repeat retrotransposable elements integrate into a precise location in the 28S rRNA genes of arthropods. The purified protein encoded by R2 can cleave the 28S gene target site and use the 3' hydroxyl group generated by this cleavage to prime reverse transcription of its own RNA, a process called target-primed reverse transcription. An integration system is described here in which components from the R2 element of the silkmoth, Bombyx mori, are injected into the preblastoderm embryo of Drosophila melanogaster. Silkmoth R2 sequences were readily detected in the 28S rRNA genes of the surviving adults as well as in the genes of their progeny. The 3' junctions of these insertions were similar to those seen in our in vitro assays, as well as those from endogenous R2 retrotransposition events. The 5' junctions of the insertions originally contained major deletions of both R2 and 28S gene sequences, a problem overcome by the inclusion of upstream 28S gene sequences at the 5' end of the injected RNA. The resulting 5' junctions suggested a recombination event between the cDNA and the upstream target sequences. This in vivo integration system should help determine the mechanism of R2 retrotransposition and be useful as a delivery system to integrate defined DNA sequences into the rRNA genes of organisms.

Identification of the endonuclease domain encoded by R2 and other site-specific, non-long terminal repeat retrotransposable elements.

The non-long terminal repeat (LTR) retrotransposon, R2, encodes a sequence-specific endonuclease responsible for its insertion at a unique site in the 28S rRNA genes of arthropods. Although most non-LTR retrotransposons encode an apurinic-like endonuclease upstream of a common reverse transcriptase domain, R2 and many other site-specific non-LTR elements do not (CRE1 and 2, SLACS, CZAR, Dong, R4). Sequence comparison of these site-specific elements has revealed that the region downstream of their reverse transcriptase domain is conserved and shares sequence features with various prokaryotic restriction endonucleases. In particular, these non-LTR elements have a Lys/Arg-Pro-Asp-X12-14aa-Asp/Glu motif known to lie near the scissile phosphodiester bonds in the protein-DNA complexes of restriction enzymes. Site-directed mutagenesis of the R2 protein was used to provide evidence that this motif is also part of the active site of the endonuclease encoded by this element. Mutations of this motif eliminate both DNA-cleavage activities of the R2 protein: first-strand cleavage in which the exposed 3' end is used to prime reverse transcription of the RNA template and second-strand cleavage, which occurs after reverse transcription. The general organization of the R2 protein appears similar to the type IIS restriction enzyme, FokI, in which specific DNA binding is controlled by a separate domain located amino terminal to the cleavage domain. Previous phylogenetic analysis of their reverse transcriptase domains has indicated that the non-LTR elements identified here as containing restriction-like endonucleases are the oldest lineages of non-LTR elements, suggesting a scenario for the evolution of non-LTR elements.

The age and evolution of non-LTR retrotransposable elements.

A comprehensive phylogenetic analysis was conducted of non-long-terminal-repeat (non-LTR) retrotransposons based on an extended sequence alignment of their reverse transcriptase (RT) domain. The 440 amino acid positions used included a region proposed to be similar to the "thumb" of the right-handed RT structure found in retroviruses. All identified non-LTR elements could be grouped into 11 distinct clades. Using the rates of sequence change derived from studies of the vertical inheritance of R1 and R2 elements in arthropods as a comparison, we found no evidence for the horizontal transmission of non-LTR elements. Assuming vertical descent, the phylogeny suggested that non-LTR elements are as old as eukaryotes, with each of the 11 clades dating back to the Precambrian era. The analysis enabled us to propose a simple chronology for the acquisition of different enzymatic domains in the evolution of the non-LTR class of retrotransposons. The first non-LTR elements were sequence specific by virtue of a restriction-enzyme-like endonuclease located downstream of the RT domain. Evolving from this original group were elements (eight clades) that acquired an apurinic-apyrimidic endonuclease-like domain upstream of the RT domain. Finally, four of these clades have inherited an RNase H domain downstream of the RT domain. The phylogenies of the AP endonuclease and RNase H domains were also determined for this report and are consistent with the monophyletic acquisition of these domains. These studies represent the most comprehensive effort to date to trace the evolution of a major class of transposable elements.

The domain structure and retrotransposition mechanism of R2 elements are conserved throughout arthropods.

R2 elements are non-LTR retrotransposons that insert in the 28S rRNA genes of arthropods. Partial sequence data from many species have previously suggested that these elements have been vertically inherited since the origin of this phylum. Here, we compare the complete sequences of nine R2 elements selected to represent the diversity of arthropods. All of the elements exhibited a uniform structure. Identification of their conserved sequence features, combined with our biochemical studies, allows us to make the following inferences concerning the retrotransposition mechanism of R2. While all R2 elements insert into the identical sequence of the 28S gene, it is only the location of the initial nick in the target DNA that is rigidly conserved across arthropods. Variation at the R2 5' junctions suggests that cleavage of the second strand of the target site is not conserved within or between species. The extreme 5' and 3' ends of the elements themselves are also poorly conserved, consistent with a target primed reverse transcription mechanism for attachment of the 3' end and a template switch model for the attachment of the 5' end. Comparison of the approximately 1,000-aa R2 ORF reveals that it can be divided into three domains. The central 450-aa domain can be folded by homology modeling into a tertiary structure resembling the fingers, palm, and thumb subdomains of retroviral reverse transcriptases. The carboxyl terminal end of the R2 protein appears to be the endonuclease domain, while the amino-terminal end contains zinc finger and c-myb-like DNA-binding motifs.

Modular evolution of the integrase domain in the Ty3/Gypsy class of LTR retrotransposons.

A phylogenetic analysis of the Ty3/Gypsy group of retrotransposons identified a conserved domain (GPY/F) present in the integrases of several members of this group as well as of certain vertebrate retroviruses. The analysis suggested an evolutionary scheme for the acquisition and loss of the GPY/F domain as well as the acquisition of a chromodomain module in the integrase encoded by this group of elements that may direct targeting specificity in the host genome.

Conserved features at the 5 end of Drosophila R2 retrotransposable elements: implications for transcription and translation.

R2 non-LTR retrotransposable elements insert site-specifically into the 28S ribosomal genes of insects. The sequence of the 5' end of full-length R2 elements from thirteen species of Drosophila were compared. Sequences within the 5' untranslated region (5' UTR) revealed little to suggest the presence of a promoter. Protein translation initiates within the 5' UTR and requires the bypassing of a highly conserved termination codon preceding the single R2 open reading frame. This bypassing probably involves a conserved RNA secondary structure which brings a potential initiation codon into close proximity to this termination codon. The most highly conserved sequence within the 5' UTR has properties similar to internal ribosomal entry sites. Based on these findings, we propose that R2elements are co-transcribed with the 28S gene and are translated as part of a large ribosomal subunit.

Retrotransposable elements R1 and R2 in the rDNA units of Drosophila mercatorum: abnormal abdomen revisited.

R1 and R2 retrotransposable elements are stable components of the 28S rRNA genes of arthropods. While each retrotransposition event leads to incremental losses of rDNA unit expression, little is known about the selective consequences of these elements on the host genome. Previous reports suggested that in the abnormal abdomen (aa) phenotype of Drosophila mercatorum, high levels of rDNA insertions (R1) in conjunction with the under-replication locus (ur), enable the utilization of different ecological conditions via a population level shift to younger age. We have sequenced the R1 and R2 elements of D. mercatorum and show that the levels of R1- and R2-inserted rDNA units were inaccurately scored in the original studies of aa, leading to several misinterpretations. In particular, contrary to earlier reports, aa flies differentially underreplicate R1- and R2-inserted rDNA units, like other species of Drosophila. However, aa flies do not undergo the lower level of underreplication of their functional rDNA units (general underreplication) that is seen in wild-type strains. The lack of general underreplication is expected to confer a selective advantage and, thus, can be interpreted as an adaptation to overcome high levels of R1 and R2 insertions. These results allow us to reconcile some of the apparently contradictory effects of aa and the bobbed phenotype found in other species of Drosophila.

Mobile introns: retrohoming by complete reverse splicing.

A mobile bacterial group II intron can integrate into DNA by the reverse splicing into a target site of its RNA transcript, which then acts as a template for DNA synthesis by an encoded reverse transcriptase. Mobility does not require homologous recombination, which has important practical and evolutionary implications.

The RTE class of non-LTR retrotransposons is widely distributed in animals and is the origin of many SINEs.

RTE-1 is a non-long-terminal-repeat (non-LTR) retrotransposable element first found in the Caenorhabditis elegans genome. It encodes a 1,024-amino-acid open reading frame (ORF) containing both apurinic-apyrimidic endonuclease and reverse-transcriptase domains. A possible first ORF of only 43 amino acids overlaps with the larger ORF and may be the site of translation initiation. Database searches and phylogenetic analysis indicate that representatives of the RTE clade of non-LTR retrotransposons are found in the bovine and sheep genomes of mammals and in the silkmoth and mosquito genomes of insects. In addition, the previously identified SINEs, Art2 and Pst, from ruminate and viper genomes are shown to be truncated RTE-like retrotransposable elements. RTE-derived SINE elements are also found in mollusc and flatworm genomes. Members of the RTE clade are characterized by unusually short 3' untranslated regions that are predominantly composed of AT-rich trimer, tetramer, and/or pentamer repeats. This study establishes RTE as a very widespread clade of non-LTR retrotransposons. RTE represents the third distinct class of non-LTR retrotransposons in the vertebrate lineage (after Line 1 elements in mammals and CR1 elements in birds and reptiles).

RNA-induced changes in the activity of the endonuclease encoded by the R2 retrotransposable element.

R2 is a non-long terminal repeat retrotransposable element that inserts itself site specifically in the 28S rRNA genes of arthropods. The 120-kDa protein encoded by R2 has been shown to cleave one strand of the 28S gene at the target site and to use the 3' hydroxyl group generated from this nick to prime reverse transcription of its own RNA. This reaction has been termed target-primed reverse transcription (TPRT). Cleavage of the second DNA strand can occur in the presence or absence of reverse transcription but requires RNA. In this study, more sensitive in vitro assays have enabled further characterization of these reactions. R2 protein is capable of only a single round of TPRT because, once bound to the target DNA, it does not dissociate at physiological ionic strengths. Analysis of the role of RNA in the DNA cleavage reaction has revealed that the binding of RNA induces the R2 protein to form a multimeric complex. While larger complexes may form, the active component appears to be a dimer based on sedimentation studies and the change in stoichiometry of the cleavage reaction from a 1:1 ratio of protein subunit to target DNA in the absence of RNA to a 2:1 ratio of subunit to DNA target in the presence of RNA. Nonspecific RNA can also induce formation of this RNA-protein (RNP) complex, but the association of the protein with R2 RNA is stronger as revealed by its stability in 0.4 M NaCl. Finally, formation of the RNP complex gives rise to a 150-fold increase in the ability of the R2 endonuclease to find the target site. The specificity of this RNP complex is sufficiently great that it can find the 28S gene target site and conduct the TPRT reaction with total genomic DNA.