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.
Highlights
The structure and mechanism of self-cleaving RNAs and their catalytic role in various biological processes have been extensively reviewed [1]
Bioinformatics searches using the constraints on the Hepatitis delta virus (HDV) ribozyme secondary structure and sequence revealed similar ribozymes present in a wide range of organisms with many found upstream of reverse transcriptase sequences [10]
To ensure a full-length 5' junction had been recovered in each species as well as score the variation in these junctions, the 28S/R2 5' element junctions from these seven species were PCR amplified and an additional 3 to 7 clones sequenced. These sequences along with junctions published previously as well as junctions obtained from available trace archives indicated that unlike the extensive variation found with the R2 elements of Drosophila, the 5' junctions derived from jewel wasp (Nv), springtail, sow bug, and earwig were uniform (Figure 1B)
Summary
The structure and mechanism of self-cleaving RNAs and their catalytic role in various biological processes have been extensively reviewed [1]. The Hepatitis delta virus (HDV) encodes two such ribozymes with similar structures that function in its replication [2,3]. In vitro selection experiments based on the HDV ribozyme [6,7,8] and the subsequent discovery of the HDV-like CPEB3 ribozyme [9] revealed few invariant positions in this enzyme. Bioinformatics searches using the constraints on the HDV ribozyme secondary structure and sequence revealed similar ribozymes present in a wide range of organisms with many found upstream of reverse transcriptase sequences [10]. The proximity of many of these putative ribozymes to reverse transcriptase suggested self-cleavage had a role in the lifecycle of retrotransposable elements
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