Independently derived targeting of 28S rDNA by A- and D-clade R2 retrotransposons

Abstract

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.

Randomly Detected Genetically Modified (GM) Maize (Zea mays L.) near a Transport Route Revealed a Fragile 45S rDNA Phenotype

“…Physical association of abundant transposable elements around the 45S rDNA cluster has been linked to increase in fragility of this locus [74]. This supports the possible pathway of fragile 45S rDNA expression which starts from genomic rearrangement that leads to transposable element activation [72], to insertion of transposable elements within or around the 45S rDNA locus [75–79], and ultimately the expression of fragility at this locus [74]. Another possible pathway would be from the epigenetic alterations caused by genome rearrangement [39] that lead to defective transcription and/or replication machinery [33], and finally to its expression.…”

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

“…Because the complete R2 protein also protects a region of DNA from 10-40 bp upstream of the cleavage site, the C-terminal domain of the protein was postulated to be responsible for this upstream binding (62). Recently, however, the N-terminal domain of the R2 protein from the horseshoe crab was shown to bind this upstream region (16). The horseshoe crab R2 protein differs from the B. mori R2 in having three zinc-finger domains instead of one (figure 2).…”

“…The two exceptions are the R2 elements of hydra, named R8, which insert into a specific sequence of the 18S rRNA gene (13), and the R2 elements of rotifer, named R9, which insert into a different site in the 28S rRNA gene (15). The ORF of all R2 elements is also very similar in coding capacity; the only significant difference is the number of zinc-finger motifs associated with DNA binding at the amino-terminal end of the protein (11,13,16). As described by Fujiwara in this volume (17), many other lineages of non-LTR retrotransposons have evolved sequence specificity for the rRNA genes or for other repeated sequences in the genomes of eukaryotes (18-22).…”

Site-specific non-LTR retrotransposons

“…The target recognition of longer sequence may explain a lack of target similarity between R2 and R8 (77). Recent reports by Christensen and co-workers suggest that the members of R2-A and R2-D subclades use different targeting mechanisms (85,86).…”

“…The other protein subunit bound to the 5′ UTR of the RNA is bound downstream from the insertion site through the N-terminal ZF and Myb motifs and may be involved in the second DNA-strand synthesis (86,120). Myb is a major contributor to the specificity and ZF provides some DNA contacts in R2, while other clades of RLE elements have no Myb motif (85,86,120). Recent studies suggest that R2-A clade elements, such as R2Lp and R9, may use Myb and ZF (mainly the third ZF from the N-terminal) motifs to bind to upstream sequences of the insertion site (86).…”