R2 Retrotransposons Research Articles

All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.

Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

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.

Internal initiation of reverse transcription in a Penelope-like retrotransposon

Eukaryotic retroelements are generally divided into two classes: long terminal repeat (LTR) retrotransposons and non-LTR retrotransposons. A third class of eukaryotic retroelement, the Penelope-like elements (PLEs), has been well-characterized bioinformatically, but relatively little is known about the transposition mechanism of these elements. PLEs share some features with the R2 retrotransposon from Bombyx mori, which uses a target-primed reverse transcription (TPRT) mechanism, but their distinct phylogeny suggests PLEs may utilize a novel mechanism of mobilization. Using protein purified from E. coli, we report unique in vitro properties of a PLE from the green anole (Anolis carolinensis), revealing mechanistic aspects not shared by other retrotransposons. We found that reverse transcription is initiated at two adjacent sites within the transposon RNA that is not homologous to the cleaved DNA, a feature that is reflected in the genomic “tail” signature shared between and unique to PLEs. Our results for the first active PLE in vitro provide a starting point for understanding PLE mobilization and biology.

Distinct and overlapping RNA determinants for binding and target-primed reverse transcription by Bombyx mori R2 retrotransposon protein.

Eukaryotic retrotransposons encode a reverse transcriptase that binds RNA to template DNA synthesis. The ancestral non-long terminal repeat (non-LTR) retrotransposons encode a protein that performs target-primed reverse transcription (TPRT), in which the nicked genomic target site initiates complementary DNA (cDNA)synthesis directly into the genome. The best understood model system for biochemical studies of TPRT is the R2 protein from the silk moth Bombyx mori. The R2 protein selectively binds the 3' untranslated region of its encoding RNA as template for DNA insertion to its target site in 28S ribosomal DNA. Here, binding and TPRT assays define RNA contributions to RNA-protein interaction, template use for TPRT and the fidelity of template positioning for TPRT cDNA synthesis. We quantify both sequence and structure contributions to protein-RNA interaction. RNA determinants of binding affinity overlap but are not equivalent to RNA features required for TPRT and its fidelity of template positioning for full-length TPRT cDNA synthesis. Additionally, we show that a previously implicated RNA-binding protein surface of R2 protein makes RNA binding affinity dependent on the presence of two stem-loops. Our findings inform evolutionary relationships across R2 retrotransposon RNAs and are a step toward understanding the mechanism and template specificity of non-LTR retrotransposon mobility.

Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon

Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.

Impaired function of rDNA transcription initiation machinery leads to derepression of ribosomal genes with insertions of R2 retrotransposon.

Eukaryotic genomes harbor hundreds of rRNA genes, many of which are transcriptionally silent. However, little is known about selective regulation of individual rDNA units. In Drosophila melanogaster, some rDNA repeats contain insertions of the R2 retrotransposon, which is capable to be transcribed only as part of pre-rRNA molecules. rDNA units with R2 insertions are usually inactivated, although R2 expression may be beneficial in cells with decreased rDNA copy number. Here we found that R2-inserted rDNA units are enriched with HP1a and H3K9me3 repressive mark, whereas disruption of the heterochromatin components slightly affects their silencing in ovarian germ cells. Surprisingly, we observed a dramatic upregulation of R2-inserted rRNA genes in ovaries lacking Udd (Under-developed) or other subunits (TAF1b and TAF1c-like) of the SL1-like complex, which is homologues to mammalian Selective factor 1 (SL1) involved in rDNA transcription initiation. Derepression of rRNA genes with R2 insertions was accompanied by a reduction of H3K9me3 and HP1a enrichment. We suggest that the impairment of the SL1-like complex affects a mechanism of selective activation of intact rDNA units which competes with heterochromatin formation. We also propose that R2 derepression may serve as an adaptive response to compromised rRNA synthesis.

R2 and Non-Site-Specific R2-Like Retrotransposons of the German Cockroach, Blattella germanica.

The structural and functional organization of the ribosomal RNA gene cluster and the full-length R2 non-LTR retrotransposon (integrated into a specific site of 28S ribosomal RNA genes) of the German cockroach, Blattella germanica, is described. A partial sequence of the R2 retrotransposon of the cockroach Rhyparobia maderae is also analyzed. The analysis of previously published next-generation sequencing data from the B. germanica genome reveals a new type of retrotransposon closely related to R2 retrotransposons but with a random distribution in the genome. Phylogenetic analysis reveals that these newly described retrotransposons form a separate clade. It is shown that proteins corresponding to the open reading frames of newly described retrotransposons exhibit unequal structural domains. Within these retrotransposons, a recombination event is described. New mechanism of transposition activity is discussed. The essential structural features of R2 retrotransposons are conserved in cockroaches and are typical of previously described R2 retrotransposons. However, the investigation of the number and frequency of 5′-truncated R2 retrotransposon insertion variants in eight B. germanica populations suggests recent mobile element activity. It is shown that the pattern of 5′-truncated R2 retrotransposon copies can be an informative molecular genetic marker for revealing genetic distances between insect populations.

Characterization and Evolution of Germ1, an Element that Undergoes Diminution in Lampreys (Cyclostomata: Petromyzontidae).

The sea lamprey (Petromyzon marinus) undergoes substantial genomic alterations during embryogenesis in which specific sequences are deleted from the genome of somatic cells yet retained in cells of the germ line. One element that undergoes diminution in P. marinus is Germ1, which consists of a somatically rare (SR) region and a fragment of 28S rDNA. Although the SR-region has been used as a marker for genomic alterations in lampreys, the evolutionary significance of its diminution is unknown. We examined the Germ1 element in five additional species of lamprey to better understand its evolutionary significance. Each representative species contained sequences similar enough to the Germ1 element of P. marinus to be detected via PCR and Southern hybridizations, although the SR-regions of Lampetra aepyptera and Lethenteron appendix are quite divergent from the homologous sequences of Petromyzon and three species of Ichthyomyzon. Lamprey Germ1 sequences have a number of features characteristic of the R2 retrotransposon, a mobile element that specifically targets 28S rDNA. Phylogenetic analyses of the SR-regions revealed patterns generally consistent with relationships among the species included in our study, although the 28S-fragments of each species/genus were most closely related to its own functional rDNA, suggesting that the two components of Germ1 were assembled independently in each lineage. Southern hybridizations showed evidence of genomic alterations involving Germ1 in each species. Our results suggest that Germ1 is a R2 retroelement that occurs in the genome of P. marinus and other petromyzontid lampreys, and that its diminution is incidental to the reduction in rDNA copies during embryogenesis.

The nucleolar transcriptome regulates Piwi shuttling between the nucleolus and the nucleoplasm.

The nucleolus contains a lot of proteins unrelated to ribosome biogenesis. Some of these proteins shuttle between the nucleolus and the nucleoplasm regulating the cell cycle and stress response. The piRNA binding protein Piwi is involved in silencing of transposable elements (TEs) in the Drosophila gonads. Here we used cultured ovarian somatic cells (OSC) to characterize Piwi as a visitor to the nucleolus. Dynamic Piwi localization was shown to vary from its uniform distribution between the nucleoplasm and the nucleolus to pronounced nucleolar immobilization. We were intrigued by this localization behavior and revealed that nascent nucleolar transcripts recruit Piwi for nucleolar retention. Piwi eviction from the nucleolus was observed upon RNase treatment and after RNA polymerase (Pol) I inhibition, but not after Pol II inactivation. On the contrary, heat shock caused drastic Piwi redistribution from the nucleoplasm to the nucleolus, which occurred only in the presence of Pol I-mediated transcription. These results allow us to hypothesizethat specific stress-induced transcripts made by Pol I promote the nucleolar sequestration of proteins in Drosophila, similar to previous observations in mammalian cells. We also found that in OSC, Piwi partially restricts expression of the rDNA copies containing R1 and R2 retrotransposon insertions especially upon heat shock-induced activation of these copies. Therefore, we suggest that Piwi intranuclear shuttling may have a functional role in ensuring a balance between silencing of rDNA-specific TEs under stress and the canonical Piwi function in non-nucleolar TE repression.

Inheritance of 5'-Truncated Copies of R2 Retrotransposon in a Series of Generations of German Cockroach, Blattella germanica

Inheritance of 5'-truncated copies of R2 retrotransposon of German cockroach, Blattella germanica, was analyzed in a series of generations. It was shown that copies of certain length are linked and form different patterns associated with X chromosomes where R2 retrotransposons are normally localized within clusters of ribosomal genes and, unexpectedly with autosomes. There was no recombination between certain copies of different length, and new variants of 5'-truncated copies were not found. Taking into account the stable inheritance of particular patterns of various R2 5'-truncated copies, we suggest using them as convenient molecular markers in population genetics studies of German cockroach, Blattella germanica. It was also shown that only short degenerate DNA sequences of R2 retrotransposon were present outside the cluster of ribosomal genes; apparently, DNA sequences corresponding to native full-length copies and/or open reading frames of R2 retrotransposon are present only within the cluster.

R1 retrotransposons in the nucleolar organizers of Drosophila melanogaster are transcribed by RNA polymerase I upon heat shock

ABSTRACTThe ribosomal RNA genes (rDNA) of Drosophila melanogaster reside within centromere-proximal nucleolar organizers on both the X and Y chromosomes. Each locus contains between 200–300 tandem repeat rDNA units that encode 18S, 5.8S, 2S, and 28S ribosomal RNAs (rRNAs) necessary for ribosome biogenesis. In arthropods like Drosophila, about 60% of the rDNA genes have R1 and/or R2 retrotransposons inserted at specific sites within their 28S regions; these units likely fail to produce functional 28S rRNA. We showed earlier that R2 expression increases upon nucleolar stress caused by the loss of the ribosome assembly factor, Nucleolar Phosphoprotein of 140 kDa (Nopp140). Here we show that R1 expression is selectively induced by heat shock. Actinomycin D, but not α-amanitin, blocked R1 expression in S2 cells upon heat shock, indicating that R1 elements are transcribed by Pol I. A series of RT-PCRs established read-through transcription by Pol I from the 28S gene region into R1. Sequencing the RT-PCR products confirmed the 28S-R1 RNA junction and the expression of R1 elements within nucleolar rDNA rather than R1 elements known to reside in centromeric heterochromatin. Using a genome-wide precision run-on sequencing (PRO-seq) data set available at NCBI-GEO, we show that Pol I activity on R1 elements is negligible under normal non-heat shock conditions but increases upon heat shock. We propose that prior to heat shock Pol I pauses within the 5ʹ end of R1 where we find a consensus “pause button”, and that heat shock releases Pol I for read-through transcription farther into R1.

Hybridogenesis and a potential case of R2 non-LTR retrotransposon horizontal transmission in Bacillus stick insects (Insecta Phasmida)

Horizontal transfer (HT) is an event in which the genetic material is transferred from one species to another, even if distantly related, and it has been demonstrated as a possible essential part of the lifecycle of transposable elements (TEs). However, previous studies on the non-LTR R2 retrotransposon, a metazoan-wide distributed element, indicated its vertical transmission since the Radiata-Bilateria split. Here we present the first possible instances of R2 HT in stick insects of the genus Bacillus (Phasmida). Six R2 elements were characterized in the strictly bisexual subspecies B. grandii grandii, B. grandii benazzii and B. grandii maretimi and in the obligatory parthenogenetic taxon B. atticus. These elements were compared with those previously retrieved in the facultative parthenogenetic species B. rossius. Phylogenetic inconsistencies between element and host taxa, and age versus divergence analyses agree and support at least two HT events. These HT events can be explained by taking into consideration the complex Bacillus reproductive biology, which includes also hybridogenesis, gynogenesis and androgenesis. Through these non-canonical reproductive modes, R2 elements may have been transferred between Bacillus genomes. Our data suggest, therefore, a possible role of hybridization for TEs survival and the consequent reshaping of involved genomes.

Participation of the piRNA pathway in recruiting a component of RNA polymerase I transcription initiation complex to germline cell nucleoli

Proteins of the Piwi family and short Piwi-interacting RNAs (piRNAs) ensure the protection of the genome from transposable elements. We have previously shown that nuclear Piwi protein tends to concentrate in the nucleoli of the cells of Drosophila melanogaster ovaries. It could be hypothesized that the function of Piwi in the nucleolus is associated with the repression of R1 and R2 retrotransposons inserted into the rDNA cluster. Here, we show that Piwi participates in recruiting Udd protein to nucleoli. Udd is a component of the conserved Selectivity Factor I-like (SL1-like) complex, which is required for transcription initiation by RNA polymerase I. We found that Udd localization depends on Piwi in germline cells, but not in somatic cells of the ovaries. In contrast, knockdowns of the SL1-like components (Udd or TAF1b) do not disrupt Piwi localization. We also observed that the absence of Udd or TAF1b in germline cells, as well as the impairment of Piwi nuclear localization lead to the accumulation of late stage egg chambers in the ovaries, which could be explained by reduced rRNA transcription. These results allow us to propose for the first time a role for Piwi in the nucleolus that is not directly associated with transposable element repression.

Integration, Regulation, and Long-Term Stability of R2 Retrotransposons.

R2 elements are sequence specific non-LTR retrotransposons that exclusively insert in the 28S rRNA genes of animals. R2s encode an endonuclease that cleaves the insertion site and a reverse transcriptase that uses the cleaved DNA to prime reverse transcription of the R2 transcript, a process termed target primed reverse transcription. Additional unusual properties of the reverse transcriptase as well as DNA and RNA binding domains of the R2 encoded protein have been characterized. R2 expression is through co-transcription with the 28S gene and self-cleavage by a ribozyme encoded at the R2 5' end. Studies in laboratory stocks and natural populations of Drosophila suggest that R2 expression is tied to the distribution of R2-inserted units within the rDNA locus. Most individuals have no R2 expression because only a small fraction of their rRNA genes need to be active, and a contiguous region of the locus free of R2 insertions can be selected for activation. However, if the R2-free region is not large enough to produce sufficient rRNA, flanking units - including those inserted with R2 - must be activated. Finally, R2 copies rapidly turnover within the rDNA locus, yet R2 has been vertically maintained in animal lineages for hundreds of millions of years. The key to this stability is R2's ability to remain dormant in rDNA units outside the transcribed regions for generations until the stochastic nature of the crossovers that drive the concerted evolution of the rDNA locus inevitably reshuffle the inserted and uninserted units, resulting in transcription of the R2-inserted units.

Deletion of Drosophila Nopp140 induces subcellular ribosomopathies.

The nucleolar and Cajal body phosphoprotein of 140kDa (Nopp140) is considered a ribosome assembly factor, but its precise functions remain unknown. To approach this problem, we deleted the Nopp140 gene in Drosophila using FLP-FRT recombination. Genomic PCR, reverse transcriptase-PCR (RT-PCR), and immunofluorescence microscopy confirmed the loss of Nopp140, its messenger RNA (mRNA), and protein products from all tissues examined. Nopp140-/- larvae arrested in the second instar stage and most died within 8days. While nucleoli appeared intact in Nopp140-/- cells, the C/D small nucleolar ribonucleoprotein (snoRNP) methyltransferase, fibrillarin, redistributed to the nucleoplasm in variable amounts depending on the cell type; RT-PCRs showed that 2'-O-methylation of ribosomal RNA (rRNA) in Nopp140-/- cells was reduced at select sites within both the 18S and 28S rRNAs. Ultrastructural analysis showed that Nopp140-/- cells were deficient in cytoplasmic ribosomes, but instead contained abnormal electron-dense cytoplasmic granules. Immunoblot analysis showed a loss of RpL34, and metabolic labeling showed a significant drop in protein translation, supporting the loss of functional ribosomes. Northern blots showed that pre-RNA cleavage pathways were generally unaffected by the loss of Nopp140, but that R2 retrotransposons that naturally reside within the 28S region of normally silent heterochromatic Drosophila ribosomal DNA (rDNA) genes were selectively expressed in Nopp140-/- larvae. Unlike copia elements and the related R1 retrotransposon, R2 expression appeared to be preferentially dependent on the loss of Nopp140 and not on environmental stresses. We believe the phenotypes described here define novel intracellular ribosomopathies resulting from the loss of Nopp140.

Evolution of the R2 Retrotransposon Ribozyme and Its Self-Cleavage Site

R2 is a non-long terminal repeat retrotransposon that inserts site-specifically in the tandem 28S rRNA genes of many animals. Previously, R2 RNA from various species of Drosophila was shown to self-cleave from the 28S rRNA/R2 co-transcript by a hepatitis D virus (HDV)-like ribozyme encoded at its 5' end. RNA cleavage was at the precise 5' junction of the element with the 28S gene. Here we report that RNAs encompassing the 5' ends of R2 elements from throughout its species range fold into HDV-like ribozymes. In vitro assays of RNA self-cleavage conducted in many R2 lineages confirmed activity. For many R2s, RNA self-cleavage was not at the 5' end of the element but at 28S rRNA sequences up to 36 nucleotides upstream of the junction. The location of cleavage correlated well with the types of endogenous R2 5' junctions from different species. R2 5' junctions were uniform for most R2s in which RNA cleavage was upstream in the rRNA sequences. The 28S sequences remaining on the first DNA strand synthesized during retrotransposition are postulated to anneal to the target site and uniformly prime second strand DNA synthesis. In species where RNA cleavage occurred at the R2 5' end, the 5' junctions were variable. This junction variation is postulated to result from the priming of second strand DNA synthesis by chance microhomologies between the target site and the first DNA strand. Finally, features of R2 ribozyme evolution, especially changes in cleavage site and convergence on the same active site sequences, are discussed.

A Population Genetic Model for the Maintenance of R2 Retrotransposons in rRNA Gene Loci

R2 retrotransposable elements exclusively insert into the tandemly repeated rRNA genes, the rDNA loci, of their animal hosts. R2 elements form stable long-term associations with their host, in which all individuals in a population contain many potentially active copies, but only a fraction of these individuals show active R2 retrotransposition. Previous studies have found that R2 RNA transcripts are processed from a 28S co-transcript and that the likelihood of R2-inserted units being transcribed is dependent upon their distribution within the rDNA locus. Here we analyze the rDNA locus and R2 elements from nearly 100 R2-active and R2-inactive individuals from natural populations of Drosophila simulans. Along with previous findings concerning the structure and expression of the rDNA loci, these data were incorporated into computer simulations to model the crossover events that give rise to the concerted evolution of the rRNA genes. The simulations that best reproduce the population data assume that only about 40 rDNA units out of the over 200 total units are actively transcribed and that these transcribed units are clustered in a single region of the locus. In the model, the host establishes this transcription domain at each generation in the region with the fewest R2 insertions. Only if the host cannot avoid R2 insertions within this 40-unit domain are R2 elements active in that generation. The simulations also require that most crossover events in the locus occur in the transcription domain in order to explain the empirical observation that R2 elements are seldom duplicated by crossover events. Thus the key to the long-term stability of R2 elements is the stochastic nature of the crossover events within the rDNA locus, and the inevitable expansions and contractions that introduce and remove R2-inserted units from the transcriptionally active domain.

Endonuclease domain of the Drosophila melanogaster R2 non-LTR retrotransposon and related retroelements: a new model for transposition

The molecular mechanisms of the transposition of non-long terminal repeat (non-LTR) retrotransposons are not well understood; the key questions of how the 3′-ends of cDNA copies integrate and how site-specific integration occurs remain unresolved. Integration depends on properties of the endonuclease (EN) domain of retrotransposons. Using the EN domain of the Drosophila R2 retrotransposon as a model for other, closely related non-LTR retrotransposons, we investigated the EN domain and found that it resembles archaeal Holliday-junction resolvases. We suggest that these non-LTR retrotransposons are co-transcribed with the host transcript. Combined with the proposed resolvase activity of the EN domain, this model yields a novel mechanism for site-specific retrotransposition within this class of retrotransposons, with resolution proceeding via a Holliday junction intermediate.

The R2 retrotransposon RNA families

Analysis of the R2 retrotransposons from multiple silkmoth and fruitfly species have revealed three segments that contain conserved RNA secondary structures. These conserved structures play important roles in the propagation of the R2 element, including R2 RNA processing and transposon integration into the host genome as well as a likely role in translation. Two of the structured regions comprise protein binding sites: one is located in the 3' UTR and the other is in the 5' UTR close to the putative start of the R2 open reading frame (ORF). The 3' structure was deduced from chemical mapping and sequence comparison. The 5' structure was determined using a combination of chemical mapping, oligonucleotide binding, NMR and sequence analysis and contains an unusual pseudoknot structure. The third structure occurs at the 5' end of the R2 RNA and is responsible for self-cleavage of the 5' end of the element from a 28S ribosomal RNA co-transcript. A structure for this fragment was proposed based on motif searching and sequence comparison. There is remarkable similarity in sequence and structure to the hepatitis delta virus (HDV) ribozyme. Seed alignments for the 5' structure and the R2 ribozyme, containing representative sequences and consensus structures, have been submitted to the Rfam database.

Independently derived targeting of 28S rDNA by A- and D-clade R2 retrotransposons: Plasticity of integration mechanism.

Restriction-like endonuclease (RLE) bearing non-LTR retrotransposons are site-specific elements that integrate into the genome through a target primed reverse transcription mechanism (TPRT). R2 elements have been used as a model system for investigating non-LTR retrotransposon integration. We previously demonstrated that R2 retrotransposons require two subunits of the element-encoded multifunctional protein to integrate-one subunit bound upstream of the insertion site and one bound downstream. R2 elements have been phylogenetically categorized into four clades: R2-A, B, C and D, that diverged from a common ancestor more than 850 million years ago. All R2 elements target the same sequence within 28S rDNA. The amino-terminal domain of R2Bm, an R2-D clade element, contains a single zinc finger and a Myb motif that are responsible for binding R2 protein downstream of the insertion site. Target site recognition is of interest as it is the first step in the integration reaction and may help elucidate evolutionary history and integration mechanism. The amino-terminal domain of R2-A clade members contains three zinc fingers and a Myb motif. We show here that R2Lp, an R2-A clade member, uses its amino-terminal DNA binding motifs to bind upstream of the insertion site. Because the R2-A and R2-D clade elements recognize 28S rDNA differently, we conclude the A- and D-clades represent independent targeting events to the 28S site. Our results also indicate a certain plasticity of insertional mechanics exists between the two clades.