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

Abstract

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.

L1 integration in a transgenic mouse model

“…Analogous to our results, multiple successive jumps of R2 RT can occur (Bibillo and Eickbush 2002b). RT template jumping was proposed to facilitate attachment of the 5Ј end of R2 to the host DNA during integration (George et al 1996;Burke et al 1999;Eickbush et al 2000;Bibillo and Eickbush 2002b), and our data suggest that template jumping may also be a feature of L1 retrotransposition. A similar mechanism was postulated to create chimeric L1 insertions (e.g., U6-L1) (Hayward et al 1997;Buzdin et al 2002Buzdin et al , 2003Gilbert et al 2005).…”

Secondary structure models of the 3′ untranslated regions of diverse R2 RNAs

“…The 3Ј UTR sequences of R2 elements from another 28 arthropod species are now available (Lathe and Eickbush 1997;Burke et al 1999). Here, we demonstrate that the R2 protein from B. mori can use in the TPRT reaction R2 RNA from even the most evolutionarily divergent arthropod.…”

“…1B, lane 6). The 3Ј UTRs of these other R2 elements are shorter than that of R2Bm, and the R2Dmer and R2Fa elements end in a poly(A) tail (Lathe and Eickbush 1997;Burke et al 1999). The three distant R2 RNAs supported TPRT from the cleavage site (bands at ∼310 nt), TPRT from the 3Ј end of the upper strand (bands at ∼360 nt), and template jumping to a second RNA (bands at ∼590 nt).…”

R2 dynamics in Triops cancriformis (Bosc, 1801) (Crustacea, Branchiopoda, Notostraca): turnover rate and 28S concerted evolution

“…The R2 element in T. cancriformis To construct the complete sequence, six clones containing the whole insertion site at the 5 0 terminus (5 0 -TTAAkGG TAGC-3 0 ; Burke et al, 1999;Kojima and Fujiwara, 2005) Figure 1 Graphic representation of the regions sequenced for the 28S analyses, with corresponding primer pairs (listed in Table 2). The primer RIN5 was specifically used for individuals M6 and M9, which lack the complete element (see text).…”

“…In T. cancriformis, a four base deletion (TTAA) was found in R2-inserted 28S, whereas in the Drosophila genus 28S deletions are larger. On the other hand, the 3 0 end of R2Tc is congruent with that of Drosophila spp., with the typical poly-A tail and the deletion of the two Gs at the insertion site (George et al, 1995;Burke et al, 1999;Pérez-Gonzalez and Eickbush, 2001). These features are determined by the target primed reversed transcription mechanism, in the phase of DNA cleavage and cDNA synthesis (Christensen et al, 2006).…”