- Open Access
R2 and R2/R1 hybrid non-autonomous retrotransposons derived by internal deletions of full-length elements
© Eickbush and Eickbush; licensee BioMed Central Ltd. 2012
Received: 27 January 2012
Accepted: 23 May 2012
Published: 23 May 2012
R2 is a non-long terminal repeat (non-LTR) retrotransposable element that inserts site specifically into the 28S genes of the ribosomal (r)RNA gene loci. Encoded at the 5' end is a ribozyme that generates the precise 5' end by self-cleavage of a 28S gene cotranscript. Sequences at the 3' end are necessary for the R2 protein to bind RNA and initiate the target primed reverse transcription (TPRT) reaction. These minimal RNA requirements suggested that if recombination/DNA repair conjoined the 5' and 3' ends of R2, the result would be a non-autonomous element that could survive as long as autonomous R2 elements supplied the TPRT activity.
A PCR-based survey of 39 Drosophila species aided by genomic sequences from 12 of these species revealed two types of non-autonomous elements. We call these elements SIDEs (for ‘Short Internally Deleted Elements’). The first consisted of a 5' ribozyme and a 3' end of an R2 element as predicted. Variation at the 5' junctions of the R2 SIDE copies was typical for R2 insertions suggesting their propagation by TPRT. The second class of SIDE contained sequences from R1 elements, another non-LTR retrotransposon that inserts into rRNA gene loci. These insertions had an R2 ribozyme immediately upstream of R1 3' end sequences. These hybrid SIDEs were inserted at the R1 site with 14 bp target site duplications typical of R1 insertions suggesting they used the R1 machinery for retrotransposition. Finally, the survey revealed examples of U12 small nuclear (sn)RNA and tRNA sequences at the 5' end of R2 elements suggesting the R2 reverse transcriptase can template jump from the R2 transcript to a second RNA during TPRT.
The R2 SIDE and R2/R1 hybrid SIDEs are rare examples of non-autonomous retrotransposons in the Drosophila genome. Associated non-autonomous elements and in vivo template jumps are two additional characteristics R2 shares with other non-LTR retrotransposons such as mammalian L1s. Analysis of the hybrid SIDEs provides supporting evidence that R1 elements, like R2 elements, recognize their 3' untranslated region (UTR) sequences and, thus, belong to the stringent class of non-LTR elements.
The genomes of all eukaryotes contain examples of transposable elements, sequences that generally appear to be genomic parasites although such sequences are occasionally co-opted for the host's benefit [1, 2]. These mobile elements fall into families that differ in basic structure and method of transposition [3, 4]. Non-long terminal repeat (non-LTR) retrotransposable elements comprise one of the two major families of mobile elements whose movement requires reverse transcriptase. Their mechanism of integration is different from retrotransposable elements with long terminal repeats in that they use the 3' hydroxyl group at a DNA break to prime reverse transcription of their RNA transcripts; a process termed target primed reverse transcription (TPRT) . Full-length non-LTR elements encode the critical enzymes necessary for generating additional copies in the genome and are, therefore, autonomous. A common occurrence with non-LTR elements is that their insertion machinery is hijacked. The elements that parasitize the retrotransposition machinery of autonomous LINEs (for ‘Long INterspersed Elements’) have been called SINEs (for ‘Short INterspersed Elements’). They are represented by Alu elements in primates although dozens of SINE families have been found in other eukaryotic genomes [6–8]. Several SINEs were in part derived from 7SL RNA; however, with the additional exception of a SINE derived from 5 S ribosomal RNA in zebrafish , the majority of SINEs in eukaryotic genomes are derived from tRNA genes [6, 10]. While their structure is variable, the characteristic attribute of SINEs is that they are transcribed by RNA polymerase III. Recognition of the SINE transcripts by LINE proteins is necessary for their reverse transcription and insertion into a new site. This is accomplished either by sequence identity at the 3' end between the LINE and its associated SINE (stringent elements) or a less strict recognition of a simple sequence, frequently a poly(A) tail, (relaxed elements) [11–14].
While there is no direct evidence, the presence of R1 lineages outside the 28S gene (for example, telomeres) suggests R1 encodes its own promoter [23, 24]. R2 elements, however, depend on an encoded self-cleaving ribozyme at their 5' end to process the R2 transcript from a 28S cotranscript. The R2 ribozyme shows remarkable similarity to the structure of the hepatitis delta virus (HDV) ribozyme with many of the conserved nucleotides in Drosophila R2 ribozymes identical to residues in the catalytic region of the HDV ribozyme .
In our survey of the R2 ribozyme in different species of Drosophila, insertions bearing the R2 ribozyme were discovered that did not correspond to the R2 elements of that species. Here we report the discovery of non-autonomous elements with sequence identity to R2 elements as well as multiple examples of hybrid non-autonomous elements with sequence identity to both R2 and R1 elements. Because these elements are not transcribed by polymerase III and therefore not SINEs [6–8], they are referred to as SIDEs (for ‘Short Internally Deleted Elements’). Based on the divergence of their sequence and their abundance, these SIDEs appear active and have persisted for millions of years. Finally, we report evidence for template jumps in vivo to small, stable RNAs in the cell, which in one case may have established a new R2 subfamily.
D. willistoni was one of the species chosen for the 12 Drosophila genomes project , thus sequencing reads containing copies of the R2Dwi_SIDE could be obtained from the trace archive. Approximately 70 original reads corresponding to the R2 SIDE were analyzed and found to have minor 5' junction variation and less than 3% nucleotide divergence. As previously documented for R2 element junctions in many Drosophila species, most full-length R2 elements in D. willistoni insert precisely into the 28S gene. This canonical 5' junction sequence is indicated by an asterisk in the upper portion of Figure 2B. However, many D. willistoni R2 element 5' junctions have deletions of the upstream 28S sequences and/or non-templated nucleotide additions. Typical examples of the range of variation are presented below the canonical junction. The full-length R2 SIDE insertions were also found to have a precise, canonical junction and the same range of sequence variation found for the R2 elements. This variation in the 5' junctions as well as variation in the length of the poly (A) tail at the 3' end (13 to 41 A’s for R2; 14 to 38 A’s for the SIDE) suggest that the R2 SIDE in D. willistoni is actively using the retrotransposition machinery provided by the autonomous R2 element.
To determine the relative abundance of R2 and R2 SIDE, a PCR primer with sequence identity to both D. willistoni elements was used in conjunction with an upstream 28S primer. The R2 element and R2 SIDE products could be differentiated after PCR amplification because the R2 SIDE sequences contain a Bam HI restriction site. The PCR results are shown in Figure 2C. The similar intensities of the 130 bp R2 element product and the 97 bp R2 SIDE product after Bam HI digestion indicated that they are present in the D. willistoni rDNA loci in equal numbers. The unexpected 200 bp PCR product suggested an abundant third element type bearing the R2 ribozyme was also present in the 28S gene at or near the R2 site. The trace archive was searched for the origin of this product. Surprisingly, an element was found with sequence identity to both the R2 ribozyme and the 3' end of the R1 element, forming what appeared to be an R2/R1 hybrid SIDE (R2/R1Dwi_SIDE). A discussion of the R1 component of this hybrid SIDE, which is more abundant than either the full-length R2 or R2Dwi_SIDE, is presented below.
Survey for additional SIDEs
Several PCR-based surveys were performed to look for additional SIDEs containing the R2 ribozyme in other Drosophila species. First, primers 1 and 2 (Figure 1B) gave rise in most of the 39 Drosophila species analyzed to a PCR product greater than 3 kb in length consistent with the presence of full-length R2 elements; however, no additional R2 SIDEs were detected. Second, a reverse primer to the catalytic region of the ribozyme was used in conjunction with a primer to 28S sequences upstream of the R2 site to look for PCR products distinct from the full-length R2 product (Figure 1B, primers 3 and 4). This survey also did not reveal additional R2 SIDEs but did lead to the discovery of several examples of in vivo template jumps to small cellular RNAs (discussed below). These results suggest R2 SIDEs are not common in Drosophila.
A third survey was performed to look for additional hybrid SIDEs in the R1 site of Drosophila. Primer 3 was paired with a 28S primer corresponding to sequences between the R2 and R1 sites (Figure 1B, primer 5). This primer pair will only amplify R2 sequences inserted downstream of the R2 site (for example, the R1 site) . PCR products containing R2 sequences were obtained from 11 species. Sequence analysis of the products from eight species suggested that they arose from R2 insertions containing target site duplications greater than 20 bp in length, therefore, only appeared inserted downstream of the R2 site. Such target site duplications have been previously detected for R2 elements . However, an analysis of the products from Drosophila falleni Drosophila innubila and Drosophila immigrans did reveal additional SIDE elements. The 3' end of each of these insertions was obtained using a species-specific primer paired with a primer downstream of the R1 site (Figure 1B, primer 6 and primer 7).
A common property of the R1 elements in many Drosophila species, including D. willistoni, is that individual 28S genes contain multiple R1 insertions. The multiple R1s are organized in a tandem array at the target site with the individual copies separated by the 14 bp 28S gene target site duplication . A search of the D. willistoni trace archive revealed that copies of R2/R1Dwi_SIDE were interspersed with the R1 elements in these tandem arrays. This result also strongly supports the conclusion that the hybrid SIDEs are mobilized like typical R1 elements.
PCR amplifications, similar to that in Figure 2C, were performed to estimate the relative abundance of the three hybrid SIDEs (data not shown). In D. falleni, R2/R1Dfa_SIDEs and R2 elements were present at approximately equal numbers; in D. immigrans, R2 elements outnumbered R2/R1Dim_SIDEs by a factor of 5; and in D. innubila only a few copies (less than 5) of the R2/R1Din_SIDE were detected. It should be noted that when multiple stocks from a species were sampled, R2 and R1 levels varied over a threefold to fivefold range [32, 33]. Therefore, the SIDE levels detected in any one stock should not be regarded as characteristic for the species.
In vivo template jumps
The long variant of the R2 element likely originated during reverse transcription when the R2 reverse transcriptase jumped from the 5' end of the R2 RNA to snU12 RNA. This process has been described as a template jump and has been observed in vitro for the R2 reverse transcriptase  and in vivo for human L1 retrotransposition . Unlike the reoccurring jumps to snU6 by L1 which gave rise to sequence variation [37, 38], the multiple copies of R2 in D. ambigua are probably derived from a single jump to snU12 RNA since they all contain the same 6 bp repeats. Because this long form appears more abundant than the short form, one intriguing possibility is that a template jump gave rise to a new subfamily of R2 capable of retrotransposing with the upstream snU12 sequences.
If the 170 bp extension is retrotransposing with the R2 element, RNA self-cleavage should occur upstream of the U12 sequences rather than at the R2 5' junction. The products observed in T7 in vitro transcription-cleavage reactions are shown in Figure 8C. Efficient self-cleavage only occurred at the 5' end of the R2 sequences as observed for a typical Drosophila R2 ribozyme (Figure 8C, lane 1; Figure 8D, diagram 1). Two constructs were next generated in an attempt to force cleavage upstream of the U12 sequences. In the first, the two G’s at the base of the R2 P1 stem were mutated to C’s (Figure 8D, diagram 2); in the second, all but the first 12 bp of the snU12 sequence as well as the first 66 nucleotides at the 5' end of R2 were deleted (Figure 8D, diagram 3). Self-cleavage in standard in vitro reactions was not observed for either RNA construct (Figure 8C, lanes 2 and 3). We suggest the conditions needed for the self-cleavage of the R2 upstream of the snU12 extension are not met in our in vitro assay. We do not favor the alternative explanation that a single R2 insertion with U12 extension occurred in this species and was then duplicated multiple times by recombination. We have never seen high levels of amplification of a specific inserted rDNA unit in Drosophila.
Finally, two additional examples of template jumps were detected in Drosophila species. An 80 bp extension at the 5' junction of an R2 element was found in the trace archive of D. pseudoobscura (Additional file 2). These extra sequences differed at only one nucleotide position from the tRNAlys(2) of this species. The presence of the nucleotides ‘CCA’ at the 3' end of this extension, which are added to tRNA post transcriptionally, confirm that the sequence arose by a jump from the R2 RNA template to the mature tRNA. Surveying the remaining 11 Drosophila trace archives for ‘CCA’ immediately upstream of full-length R2 insertions revealed another potential template jump to tRNA in Drosophila yakuba. In this case, 18 nucleotides from tRNAgly were found at the 5' junction of an R2 (Additional file 2).
The experiments in this report provide evidence for new families of insertion elements in the 28S genes of Drosophila. Segments from R2 and/or R1 elements comprise these insertions, and they are mobilized by hijacking the R2 or R1 retrotransposition machinery. Because these non-autonomous elements rely (as does the R2 element itself) on cotranscription with the 28S gene, they are referred to as SIDEs rather than SINEs. Non-autonomous DNA-mediated transposable element families, such as the miniature inverted-repeat DINE-1 and non-autonomous P elements, have been previously documented in Drosophila genomes [39–41]. The R2 SIDE and R2/R1 hybrid SIDEs along with HeT-A  are, however, the only clear examples of non-autonomous retrotransposons to be found in Drosophila. Analysis of the SIDEs provides direct support for the model that R2 retrotransposition requires only the 5' end for RNA self-cleavage from a 28S cotranscript and the 3' UTR for binding the R2 protein to initiate TPRT. The discovery of SIDEs mobilized by the R1 machinery also provides strong support for the model  that the R1 protein recognizes the 3' UTR sequences/secondary structure of its RNA to initiate TPRT and thus belongs to the class of stringent non-LTR retrotransposable elements.
Because there is a single lineage of R2 element vertically transmitted in Drosophila, the levels of divergence between ribozyme sequences (excluding the highly variable J1/2 loop) from different elements can be compared to provide an estimate of the number of independently formed SIDEs and their approximate ages. First, the 25% sequence divergence between the ribozymes from the R2 element and R2 SIDE of D. willistoni is similar to the divergence between the ribozymes from the R2 elements from D. willistoni and D. melanogaster (23%) as well as between D. ananassae and D. melanogaste r (28%). Assuming similar levels of constraint on the ribozyme of these elements, this suggests the R2 SIDE lineage is as old as the divergence between species groups within the Sophophora subgenus, that is, over 40 million years [43, 44]. Second, the 27% sequence divergence between the R2 and hybrid SIDE ribozymes from D. immigrans indicates the R2/R1Dim_SIDE lineage also dates back to a comparable time within the Drosophila subgenus. Third, the lower levels of sequence divergence between the ribozymes from R2/R1Dwi_SIDE and R2Dwi (11%) and between the ribozymes from R2/R1Dfa_SIDE and R2Dfa (10%) suggests both of these hybrid SIDEs have a more recent origin (approximately 20 million years ago). Because D. falleni and D. willistoni are in different subgenuses, their hybrid SIDEs arose independently. Finally, because R2/R1Dfa_SIDE and R2/R1Din_SIDE have only 3% sequence divergence, they likely represent the same event in the ancestor of these two closely related species. In summary, the five identified SIDEs in this report appear to have originated in four separate events.
Non-autonomous elements of DNA transposons (for example, miniature inverted-repeat transposable elements (MITEs)) and LTR retrotransposons (for example, terminal-repeat retrotransposons in miniature (TRIMs)) have been found to originate from autonomous elements by internal deletions [6, 45–48]. The non-LTR, non-autonomous elements TbRIME and Ag-Sponge also appear to have arisen by internal deletions [49, 50]. TbRIME is of special interest because it has sequence identity at the 5' end to the ribozyme encoded by L1Tc [31, 51]. Two potential mechanisms could have formed the Drosophila SIDEs. First a template jump  during a retrotransposition reaction could have fused the 3' and 5' ends of an R2 element. The R2 5' junctions with upstream snU12 RNA and tRNA sequences shown in Figure 8 and Additional file 2 demonstrates the R2 protein does have the ability to template jump in vivo. In the case of the hybrid SIDEs, R1 sequences are located downstream of the R2 sequences, therefore, it is the R1 reverse transcriptase that must be postulated as responsible for the jumps. A second more likely possibility for the formation of the SIDEs is that non-homologous recombination within the rRNA gene locus joined the 5' end of R2 to either the 3' end of R2 or the 3' end of R1. Such recombinants could have been the result of DNA repair after retrotransposition events. The R2 machinery has been associated with large deletions of upstream rDNA sequences in D. melanogaster and D. simulans. Alternatively, the recombinations generating the SIDEs could simply have been aberrant versions of the frequent crossovers that give rise to the concerted evolution of the rDNA locus. Whatever the scenario, it seems unlikely that the SIDEs were formed in their present configuration. All SIDE families appear old, thus there has been ample opportunity for subsequent internal deletions to shorten the SIDEs until only the minimal sequences needed for activity remain.
Based on the sequence conservation of each SIDE, it appears that these elements have recently been active. Since their formation, the ribozymes and 3' ends of the SIDEs appear to be evolving similarly to the corresponding regions of R2 and R1 with two notable exceptions. A highly conserved ‘U’ located in the catalytic region of 18/19 Drosophila R2 ribozymes as well as in the R2 SIDE itself (pink circle, Figures 4A6A7A and 8D) has been substituted with an ‘A’ in all hybrid R1/R2 SIDEs. This substitution may reflect the difference in the insertion site of the hybrid SIDEs and consequently the upstream 28S sequences that must be cleaved from the cotranscripts. The second exception is a stop codon that is found in J4/2 in 18/19 R2 elements (pink box, Figures 4A6A and 8D) but not found in any of the five SIDEs. We suggest this stop codon is important in the initiation of translation of the R2 RNA open reading frame by way of an encoded internal ribosome entry site (IRES) [54, 55], a function obviously not required for RNA arising from the SIDEs.
In general, non-LTR SIDEs appear to be rare. An L1 SIDE has not been observed despite the fact that studies of L1 retrotransposition in cultured cells revealed the generation of chimeric and internally deleted L1 insertions . The cis preference of the L1 ORF2 protein for its RNA can, however, readily explain the absence of an associated SIDE . Likewise, our survey of 39 Drosophila species suggests that the formation of R2/R1 hybrid SIDEs and to a greater extent R2 SIDEs is also rare and/or their survival after formation is unlikely. While there is no evidence that R1 and R2 undergo cis preference, our studies on R2 expression and regulation suggest an explanation for the paucity of R2 associated SIDEs [57, 58]. Our current model suggests that Drosophila has the ability to select for transcription a localized region in the rDNA locus that has the lowest level of insertions. Because the SIDEs as well as the R2 elements rely on cotranscription with the 28S gene, their transcription can only occur whenever an rDNA unit with the insertion is located within this transcription domain. Consequently, in order for an R2 SIDE to retrotranspose both a copy of the SIDE and a copy of the autonomous R2 element must be present in the small transcription domain. Because the R2 lineage itself appears somewhat unstable and has been lost in several species of Drosophila[22, 59], the survival of an R2 SIDE would be even more tenuous. However, R1 elements have been suggested to contain their own promoter and thus may not need to be within the transcription domain for activity. R1 elements are present in all lineages of Drosophila and indeed many species have two distinct lineages [21, 59]. The greater evolutionary stability of the R1 retrotransposition machinery and the independence of transcriptional control of the hybrid SIDE from the autonomous R1 elements may explain why these SIDEs appear to have a greater chance of long-term survival within the locus.
This report demonstrates that R1 and R2 elements, like many other non-LTR retrotransposons, are parasitized by non-autonomous sequences that hijack their retrotransposition machinery. These short internally deleted elements, or SIDEs as we have called them, need only the R2 self-cleaving ribozyme at their 5’ end to process themselves from a 28S rRNA co-transcript and 3’ RNA sequences which can be bound by the retrotransposition machinery of an autonomous element. These R2 SIDEs and R2/R1 SIDEs can survive only as long as the autonomous R1 and R2 elements are able to survive. The existence of each element would seem tenuous, as there are a limited number of potential insertion sites in the rDNA locus. However, the high rates of recombination and turnover of rDNA units within this locus facilitates mobile element survival [20, 21, 57, 59]. The finding that some lineages of the SIDEs have persisted for an estimated 40 million years suggests this genomic niche is sometimes even flexible enough to maintain the parasites of R1 and R2.
PCR amplification/cloning/nucleotide sequence determination
Genomic DNA from most Drosophila species surveyed was previously isolated [20, 21]. For D. innubila and Drosophila phalerata, genomic DNA was isolated from adult flies (a gift from J Jaenike) as described in the above references. The initial survey for R2 SIDEs was performed using two primers to the conserved catalytic region of the R2 ribozyme (R2(catA), 5'-AAAACCTCCTCGTGGTRTY-3') and (R2(catB), 5'-GTGGCCTCCTCGTGGTRTY-3') separately paired with a reverse primer which anneals to the 28S gene 29 to 50 nucleotides downstream of the R2 site (28S(+50), 5'-CGTTAATCCATTCATGCGCGTC-3'). The survey for R2/R1 hybrid SIDEs was performed using a reverse primer to the conserved catalytic region of the R2 ribozyme (R2 (cat1), 5'-RAYACCACGAGGAGG-3') paired with a primer annealed to the 28S gene 1 to 15 nucleotides downstream of the R2 insertion site (28S (+15), 5'-TAGCCAAATGCCTCG-3'). A second survey for R2 SIDEs and R2 5' variation was performed by pairing the R2 (cat1) primer with a 28S gene primer 81 to 61 nucleotides upstream of the R2 insertion site (28S (−81), 5'-TGCCCAGTCCTCTGAATGTC-3'). Where noted R corresponds to A and G; Y corresponds to C and G; and W corresponds to A and T. PCR fragments were cloned into the pCR2.1-TOPO cloning vector (Invitrogen, Grand Island, NY USA) and sequenced (Macrogen, Rockville, MD USA).
The 3' ends of the R2/R1 SIDEs were obtained by pairing the D. falleni/D. innubila primer (fal(J1/2), 5'-GCACATGGTGTCCCACAAATTGTCAG-3') and the D. immigrans primer (imm(J1/2), 5'-TACCTTGGCAAAGTACCC-3') with a reverse primer which annealed to the 28S gene 6 to 27 nucleotides downstream of the R1 site (28S(+80), 5'-GTTCCCTTGGCTGTGGTTTCGC-3'). The 3' end of the R2 ribozyme from D. ambigua was obtained by pairing primer (Cys(amb), 5'-CATRTGNACRCCNARNCC) with (28S(−81)). A partial snU12 sequence from D. ambigua was obtained by pairing primers: (DpsU12up, 5'-GTGCCTGAAATTAATGAGTAAGG) and (DpsU12down, GGGCAGATCGCAAACACCC). All PCR products were cloned and sequenced as above.
The primers to sequences shared by the R2 element and SIDE(s) in D. willistoni (Cons(wil), 5'-ACACCACGAGGAGGTTTCG-3'), in D. falleni/D. innubila (Cons(fal), 5'-ACACTGAATTTAGCACCCGGAGG-3'), and in D. immigrans (Cons(imm), 5'-ACGGWGGCCCCCTCTGC-3') were paired with either 28S(−81) or (28S(−32), 5'-CAACGGCGGGAGTAACTATG-3') to determine relative SIDE abundance. PCR products were separated on 8.75% polyacrylamide gels and ethidium bromide stained bands analyzed using QuantityOne (BioRad, Hercules, CA USA).
Reverse primers which annealed to sequences downstream of the SIDE ribozymes: D. willistoni (R2SIDE(wil), 5'-AGGATTAGACCTTCAGAATACC-3') and (R2/R1SIDE(wil), 5'- GCCAAACAGGAAATGGGTAAACC-3') D. falleni/D. innubila (R2/R1SIDE(fal), 5'-CTACCAATTCTAACTCCAAAACAG-3'), and D. immigrans (R2/R1SIDE(imm), 5'-TATGGAAGAATTCTAACCCGC-3') as well as downstream of the R2 elements: D. willistoni (R2(wil), 5'-GGTAACCCCAAGAGTTGCTTC-3'), D. falleni/D. innubila (R2(fal), 5'-TTGGGTAGGTAACCCTTTGGAC-3'), D. immigrans (R2(imm), 5'-TGATTTGCACCAACAGTTGTC-3') and D. ambigua (R2(amb), 5'-CCCCATAGGACTGTTTCGCTG-3') were paired with a 28S upstream primer containing a T7 promoter (28S(−95), 5'-TAATACGACTCACTATAGGGCACAATGTGATTTCTGCCCAGT-3'). PCR fragments were cloned into the TOPO cloning vector (Invitrogen, Grand Island, NY USA). DNA templates were generated by PCR amplification using the same primers with unincorporated primers and nucleotides removed with the PCR Purification Kit (BioBasics, Markham, Ontario Canada).
Assays were preformed as described in . Approximately 0.1 μg of PCR template was incubated in transcription buffer with 20 units of T7 RNA polymerase (Fermentas, Glen Burnie, MD USA) and trace amounts of [α-32P]UTP) for 1 h at 42°C. Reactions were then placed on ice and four volumes of 95% formamide, 10 mM EDTA (pH 8) added. RNA products were denatured at 92°C for 3 minutes and separated on 8 M urea, 5% polyacrylamide gels. The dried gels were exposed to a phosphorimager screen and analyzed using QuantityOne (BioRad, Hercules, CA USA).
SIDE sequence files
Complete nucleotide sequences for each SIDE can be found in Additional file 3 (R2Dwi_SIDE), Additional file 4 (R2/R1Dwi_SIDE), Additional file 5 (R2/R1Dfa_SIDE), Additional file 6 (R2/R1Din_SIDE), and Additional file 7 (R2/R1Dim_SIDE). Sequences were aligned with the aid of ClustalX .
Original sequence reads
Sequencing reads from the whole genome shotgun sequencing project of D. willistoni (8.4-fold coverage), D. pseudoobscura (ninefold coverage), and D. yakuba (ninefold coverage) were accessed by Blast search (version 2.2.17) in the trace archives at NCBI .
The authors thank William Burke for helpful discussions. We thank J Jaenike (University of Rochester) for fly stocks. This work was made possible by National Institutes of Health Grant Number R01GM42790.
- Volff JN: Turning junk into gold: domestication of transposable elements and the creation of new genes in eukaryotes. Bioessays. 2006, 28: 913-922.View ArticlePubMedGoogle Scholar
- Hedges DJ, Deininger PL: Inviting instability: transposable elements, double-strand breaks, and the maintenance of genome integrity. Mutat Res. 2007, 616: 46-59.PubMed CentralView ArticlePubMedGoogle Scholar
- Craig Nl, Craige R, Gellert M, Lambowitz AM: Mobile DNA II. 2002, ASM Press, Washington, DC, USAView ArticleGoogle Scholar
- Levin HL, Moran JV: Dynamic interactions between transposable elements and their hosts. Nat Rev Genet. 2011, 12: 615-627.PubMed CentralView ArticlePubMedGoogle Scholar
- Luan DD, Korman MH, Jakubczak JL, Eickbush TH: Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell. 1993, 72: 595-605.View ArticlePubMedGoogle Scholar
- Kramerov DA, Vassetzky NS: Short retroposons in eukaryotic genomes. Int Rev Cytol. 2005, 247: 165-221.View ArticlePubMedGoogle Scholar
- Ohshima K, Okada N: SINEs and LINEs: symbionts of eukaryotic genomes with a common tail. Cytogenet Genome Res. 2005, 110: 475-490.View ArticlePubMedGoogle Scholar
- Belancio VP, Hedges DJ, Deininger P: Mammalian non-LTR retrotransposons: for better or worse, in sickness and in health. Genome Res. 2008, 18: 343-358.View ArticlePubMedGoogle Scholar
- Kapitonov VV, Jurka J: A novel class of SINE elements derived from 5 S rRNA. Mol Biol Evol. 2003, 20: 694-702.View ArticlePubMedGoogle Scholar
- Sun F-J, Fleurdépine S, Bousquet-Antonelli C, Caetano-Anollés G, Deragon J-M: Common evolutionary trends for SINE RNA structures. Trends Genet. 2007, 23: 26-33.View ArticlePubMedGoogle Scholar
- Oshima K, Hamada M, Terai Y, Okada N: The 3' ends of tRNA-derived short interspersed repetitive elements are derived from the 3' ends of long interspersed repetitive elements. Mol Cell Biol. 1996, 16: 3756-3764.View ArticleGoogle Scholar
- Kajikawa M, Okada N: LINEs mobilize SINEs in the eel through a shared 3' sequence. Cell. 2002, 111: 433-444.View ArticlePubMedGoogle Scholar
- Dewannieux M, Esnault C, Heidmann T: LINE-mediated retrotransposition of marked Alu sequences. Nat Genet. 2003, 35: 41-48.View ArticlePubMedGoogle Scholar
- Roy-Engel AM, Salem A-H, Oyeniran OO, Deininger L, Hedges DJ, Kilroy GE, Batzer MA, Deininger PL: Active Alu element "A-tails": size does matter. Genome Res. 2002, 12: 1333-1344.PubMed CentralView ArticlePubMedGoogle Scholar
- Eickbush TH: R2 and Related site-specific non-long terminal repeat retrotransposons. Mobile DNA II. Edited by: Craig NL, Craigie R, Gellert M, Lambowitz AM. 2002, ASM Press, Washington, DC, USA, 813-835.View ArticleGoogle Scholar
- Christensen SM, Ye J, Eickbush TH: RNA from the 5’ end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site. Proc Natl Acad Sci USA. 2006, 103: 17602-17607.PubMed CentralView ArticlePubMedGoogle Scholar
- Fenq Q, Shumann G, Boeke JD: Retrotransposon R1Bm endonuclease cleaves the target sequence. Proc Natl Acad Sci USA. 1998, 95: 2083-2088.View ArticleGoogle Scholar
- Maita N, Aoyagi H, Osani M, Shirakawa M, Fujiwara H: Characterization of the sequence specificity of the R1Bm endonuclease domain by structural and biochemical studies. Nucleic Acids Res. 2007, 35: 3918-3927.PubMed CentralView ArticlePubMedGoogle Scholar
- Anzai T, Osanai M, Hamada M, Fujiwara H: Functional roles of 3'-terminal structures of template RNA during in vivo retrotransposition of non-LTR retrotransposon, R1Bm. Nucleic Acids Res. 2005, 33: 1993-2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Lathe WC, Eickbush TH: A single lineage of R2 retrotransposable elements is an active, evolutionary stable component of the Drosophila rDNA locus. Mol Biol Evol. 1997, 14: 1232-1241.View ArticlePubMedGoogle Scholar
- Gentile KL, Burke WD, Eickbush TH: Multiple lineages of R1 retrotransposable elements can coexist in the rDNA loci of Drosophila. Mol Biol Evol. 2001, 18: 235-245.View ArticlePubMedGoogle Scholar
- Stage DE, Eickbush TH: Origin of nascent lineages and the mechanisms used to prime second-strand DNA synthesis in the R1 and R2 retrotransposons of Drosophila. Genome Biol. 2009, 10: R49-PubMed CentralView ArticlePubMedGoogle Scholar
- Osani-Futahashi M, Suetsugu Y, Mita K, Fujiwara H: Genome-wide screening and characterization of transposable elements and their distribution analysis in the silkworm, Bombyx mori. Insect Biochem Mol Biol. 2008, 38: 1046-1057.View ArticleGoogle Scholar
- Kojima KK, Fujiwara H: Evolution of target specificity in R1 clade non-LTR retrotransposons. Mol Biol Evol. 2003, 20: 351-361.View ArticlePubMedGoogle Scholar
- Eickbush DG, Eickbush TH: R2 retrotransposons encode a self-cleaving ribozyme for processing from an rRNA cotranscript. Mol Cell Biol. 2010, 30: 3142-3150.PubMed CentralView ArticlePubMedGoogle Scholar
- NCBI: BLAST. [http://www.ncbi.nlm.nih.gov/BLAST/] 
- Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, Markow TA, Kaufman TC, Kellis M, Gelbart W, Iyer VN, Pollard DA, Sackton TB, Larracuente AM, Singh ND, Abad JP, Abt DN, Adryan B, Aguade M, Akashi H, Anderson WW, Aquadro CF, Ardell DH, Arguello R, Artieri CG, Barbash DA, Barker D, Barsanti P, Batterham P, Batzoglou S, Drosophila 12 Genomes Consortium: Evolution of genes and genomes on the Drosophila phylogeny. Nature. 2007, 450: 203-218.View ArticlePubMedGoogle Scholar
- Mathews DH, Banerjee AR, Luan DD, Eickbush TH, Turner DH: Secondary structure model of the RNA recognized by the reverse transcriptase from the R2 retrotransposable element. RNA. 1997, 3: 1-16.PubMed CentralPubMedGoogle Scholar
- Webb C-HT, Lupták A: HDV-like self-cleaving ribozymes. RNA Biol. 2011, 8: 719-727.PubMed CentralView ArticlePubMedGoogle Scholar
- Nehdi A, Perreault J-P: Unbiased in vitro selection reveals the unique character of the self-cleaving antigenomic HDV RNA sequence. Nucleic Acids Res. 2006, 34: 584-592.PubMed CentralView ArticlePubMedGoogle Scholar
- Sánchez-Luque FJ, López MC, Macias F, Alonso C, Thomas MC: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5'end of the L1Tc retrotransposon from Trypanosoma cruzi. Nucleic Acids Res. 2011, 39: 8065-8077.PubMed CentralView ArticlePubMedGoogle Scholar
- Jakubczak JL, Zenni MK, Woodruff RC, Eickbush TH: Turnover of R1 (Type I) and R2 (Type II) retrotransposable elements in the ribosomal DNA of Drosophila melanogaster. Genetics. 1992, 131: 129-142.PubMed CentralPubMedGoogle Scholar
- Zhang X, Eickbush TH: Characterization of active R2 retrotransposition in the rDNA locus of Drosophila simulans. Genetics. 2005, 170: 195-205.PubMed CentralView ArticlePubMedGoogle Scholar
- Tarn WY, Steitz JA: A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro. Cell. 1996, 84: 801-811.View ArticlePubMedGoogle Scholar
- Bibillo A, Eickbush TH: End-to-end template jumping by the reverse transcriptase encoded by the R2 retrotransposon. J Biol Chem. 2004, 279: 14945-14953.View ArticlePubMedGoogle Scholar
- Buzdin A, Gogvadze E, Kovalskaya E, Volchkov P, Ustyugova S, Illarionova A, Fushan A, Vinogradova T, Sverdlov E: The human genome contains many types of chimeric retrogenes generated through in vivo RNA recombination. Nucleic Acids Res. 2003, 31: 4385-4390.PubMed CentralView ArticlePubMedGoogle Scholar
- Buzdin A, Ustyugova S, Gogvadze E, Vinogradova T, Lebedev Y, Sverdlov E: A new family of chimeric retrotranscripts formed by a full copy of U6 small nuclear RNA fused to the 3' terminus of L1. Genomics. 2002, 80: 402-406.View ArticlePubMedGoogle Scholar
- Gilbert N, Lutz S, Morrish TA, Moran JV: Multiple fates of L1 retrotransposition intermediates in cultures human cells. Mol Cell Biol. 2005, 25: 7780-7795.PubMed CentralView ArticlePubMedGoogle Scholar
- Kapitonov W, Jurka J: Helitrons in fruit flies. Repbase Reports. 2007, 7: 127-132.Google Scholar
- Yang HP, Barbash DA: Abundant and species-specific DINE-1 transposable elements in 12 Drosophila genomes. Genome Biol. 2008, 9: R39-PubMed CentralView ArticlePubMedGoogle Scholar
- Engels WR: A trans-acting product needed for P factor transposition in Drosophila. Science. 1984, 226: 1194-1196.View ArticlePubMedGoogle Scholar
- Pardue ML, DeBaryshe PG: Retrotransposons provide an evolutionarily robust non-telomerase mechanism to maintain telomeres. Annu Rev Genet. 2003, 37: 485-511.View ArticlePubMedGoogle Scholar
- Tamura K, Subramoanian S, Kumar S: Temporal patterns of fruit fly (Drosophila) evolution revealed by mutation clocks. Mol Biol Evol. 2004, 21: 36-44.View ArticlePubMedGoogle Scholar
- Singh ND, Larracuente AM, Sackton TB, Clark AG: Comparative genomics on the Drosophila phylogenetic tree. Annu Rev Ecol Evol Syst. 2009, 40: 459-480.View ArticleGoogle Scholar
- Jiang N, Bao Z, Zhang X, Hirochika H, Eddy SR, McCouch SR, Wessler SR: An active DNA transposon family in rice. Nature. 2003, 421: 163-167.View ArticlePubMedGoogle Scholar
- Witte C-P, Le QH, Bureau T, Kumar A: Terminal-repeat retrotransposons in miniature (TRIM) are involved in restructuring plant genomes. Proc Natl Acad Sci USA. 2001, 98: 13778-13783.PubMed CentralView ArticlePubMedGoogle Scholar
- Antonius-Klemola K, Kalendar R, Schulman AH: TRIM retrotransposons occur in apple and are polymorphic between varieties but not sports. Theor Appl Genet. 2006, 112: 999-1008.View ArticlePubMedGoogle Scholar
- Kwon SJ, Kim DH, Lim MH, Long Y, Meng JL, Lim KB, Kim JA, Kim JS, Jin M, Kim HI, Ahn SN, Wessler SR, Yang TJ, Park BS: Terminal repeat retrotransposon in miniature (TRIM) as DNA markers in Brassica relatives. Mol Genet Genomics. 2007, 278: 361-370.View ArticlePubMedGoogle Scholar
- Kimmel BE, Ole-Moiyoi OK, Young JR: Ingi, a 5.2-kb dispersed sequence element from Trypanosoma brucei that carries half of a smaller mobile element at either end and has homology with mammalian LINEs. Mol Cell Biol. 1987, 7: 1465-1475.PubMed CentralView ArticlePubMedGoogle Scholar
- Biedler J, Tu Z: Non-LTR retrotransposons in the African malaria mosquito, Anopheles gambiae: unprecedented diversity and evidence of recent activity. Mol Biol Evol. 2003, 20: 1811-1825.View ArticlePubMedGoogle Scholar
- Bringaud F, Berriman M, Fowler-Hertz C: TSIDER1, a short and non-autonomous Salivarian trypanosome-specific retroposon related to the ingi6 subclade. Mol Biochem Parasitology. 2011, 179: 30-36.View ArticleGoogle Scholar
- Eickbush DG, Eickbush TH: Transcription of endogenous and exogenous R2 elements in the rRNA gene locus of Drosophila melanogaster. Mol Cell Biol. 2003, 23: 3825-3836.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang X, Zhou J, Eickbush TH: Rapid R2 retrotransposition leads to the loss of previously inserted copies via large deletions of the rDNA locus. Mol Biol Evol. 2008, 25: 229-237.View ArticlePubMedGoogle Scholar
- George JA, Eickbush TH: Conserved features at the 5' end of Drosophila R2 retrotransposable elements: implications for transcription and translation. Insect Mol Biol. 1999, 8: 3-10.View ArticlePubMedGoogle Scholar
- Ruminski DJ, Webb C-HT, Riccitelli NJ, Lupták A: Processing and translation initiation of non-long terminal repeat retrotransposons by hepatitis delta virus (HDV)-like self-cleaving ribozymes. J Biol Chem. 2011, 286: 41286-41295.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei W, Gilbert N, Ooi SL, Lawler JF, Ostertag EM, Kazazian HH, Boeke JD, Moran JV: Human L1 retrotransposition: cis preference versus trans complementation. Mol Cell Biol. 2001, 21: 1429-1439.PubMed CentralView ArticlePubMedGoogle Scholar
- Eickbush DG, Ye J, Zhang X, Burke WD, Eickbush TH: Epigenetic regulation of retrotransposons within the nucleolus of Drosophila. Mol Cell Biol. 2008, 28: 6452-6461.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou J, Eickbush TH: The pattern of R2 retrotransposon activity in natural populations of Drosophila simulans reflects the dynamic nature of the rDNA locus. PLoS Genet. 2009, 5: e1000386-PubMed CentralView ArticlePubMedGoogle Scholar
- Eickbush DG, Eickbush TH: Vertical transmission of the retrotransposable elements R1 and R2 during the evolution of the Drosophila melanogaster species subgroup. Genetics. 1995, 139: 671-684.PubMed CentralPubMedGoogle Scholar
- Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tool. Nucleic Acids Res. 1997, 25: 4876-4882.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.