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

Abstract

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.

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

“…Consistent with this model, in all animals the junctions of 5’ truncated R2 insertions, which are generated when the reverse transcriptase doesn't reach the 5’ end of the RNA template, are found to involve microhomologies and non-templated additions (84,87,94). Direct support for this model has also been provided by in vivo R2 injection experiments in Drosophila, which yielded integrations with precise 5’ junctions when the injected R2 RNA contained upstream 28S sequences and highly variable junctions when the injected R2 RNA did not contain upstream 28S sequences (74).…”

“…An analysis of R2 from many other animals spanning its entire host range revealed that the 5’ end of all R2 elements could be folded into an HDV-like ribozyme structure (84). Comparison of these different R2 ribozymes revealed considerable flexibility in some aspects of the structure and in the sequence.…”

“…Unlike the D. simulans R2 ribozyme, which cleaves at the precise 28S/R2 5’junction, many R2 ribozymes cleaved in GC-rich regions of the 28S rRNA either 13 or 28 nucleotides upstream of the R2 insertion site (Figure 3B). As described below in the discussion of the R2 integration mechanism, self-cleavage by the ribozyme in the upstream 28S sequences in some species gives rise to insertions with uniform 5’ junctions while self-cleavage at the 5’ end of R2 in other species gives rise to variable junctions (11,84,87). …”

“…Based on the variation observed at the 5’ junction of endogenous R2 insertions in different species, it also appears to be a step that has evolved. In B. mori and many other animals, the R2 5' junctions within a species show little variation except for the occasional direct duplication of 28S gene sequences of a characteristic length for each species (11,84). However, in species of Drosophila and other animals, most 5’ junctions contain variable deletions of the 28S target site and variable additions of non-templated nucleotides (87,94).…”

Site-specific non-LTR retrotransposons

“…7B, which shows similar steps in R2Ol). The self-cleavage site in most R2 elements from different species was shown not at the 5′ end of the element but at 28S sequences up to 36 nucleotides upstream of the 5′ junction (98). It has been postulated that the cotranscribed 28S sequences of the first cDNA strand at the 5′ junction may anneal to the target site and prime the synthesis of the second DNA strand (98).…”