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. 2012 May 23;3(1):10.
doi: 10.1186/1759-8753-3-10.

R2 and R2/R1 hybrid non-autonomous retrotransposons derived by internal deletions of full-length elements

Danna G Eickbush  1 , Thomas H Eickbush
Affiliations

R2 and R2/R1 hybrid non-autonomous retrotransposons derived by internal deletions of full-length elements

Danna G Eickbush et al. Mob DNA. .

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

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Figures

Figure 1
Figure 1
The rDNA locus and its R2 and R1 element insertions. (A) The rDNA locus is composed of a tandem array of rDNA units with a subset of these units inserted by R2 (blue boxes) and/or R1 elements (orange boxes). The rRNA transcription unit with external transcribed spacer (ETS), 18S, 5.8S and 28S genes (gray boxes), transcribed spacers (white boxes), and R2 insertion is diagrammed. The single open reading frame (ORF) of R2 is delineated in light blue. R2 RNA sequences are processed from the cotranscript at the 5' end by an R2 encoded self-cleaving ribozyme. After translation, identical subunits of the R2 protein (circles) bind sequences at either end of the R2 transcript, and the RNA/protein complex binds at the R2 target site in the 28S gene and proceeds with the insertion of a new R2 copy. (B) Diagram of a portion of the 28S gene with both R2 and R1 insertions. Arrows indicate location and direction of primers in the 28S gene and R2 element used to survey for unusual insertions near the R2 target site.
Figure 2
Figure 2
R2 SIDE (‘Short Internally Deleted Element’) inDrosophila willistoni.(A) The 3.53 kb R2 element in D. willistoni, R2Dwi, is diagrammed with the 5' and 3' UTRs (untranslated regions) shaded darker. The 529 bp element, R2Dwi_SIDE, has sequence identity at the 5' and 3' ends to the R2 element (percent identity shown); the 197 bp central region (white box) has no significant identity to the R2 element. (B) Sequence reads for full-length R2 and R2Dwi_SIDE elements obtained from the trace archive at NCBI [26]. The majority of 5' junctions for both element types are precise (marked with asterisks). Typical variation at the 5' junction for both elements is also presented. (C) Genomic DNA from D. willistoni was PCR amplified using a 28S primer (32 nucleotides upstream of the R2 site) and a ribozyme primer (conserved region 100 nucleotides into the elements) (arrows). PCR products after BamHI digestion were separated on a native, 8% polyacrylamide gel. Lane M, DNA length markers with sizes indicated. The product at 200 bp was subsequently determined to correspond to an insertion in the R1 site, R2/R1Dwi_SIDE (bottom diagram). Element type and relative percentage in the genome are to the right of the gel.
Figure 3
Figure 3
Secondary structure conservation of R2 3' ends. (A) RNA sequence from the 3' UTR of the R2 element from Drosophila willistoni folded into the secondary structure modeled for other Drosophila R2 [28]. Nucleotides identical to those found to be conserved in the previous report are boxed. (B) The 3' end sequence from R2Dwi_SIDE folded into the same secondary structure. Nucleotide differences relative to R2Dwi are circled in blue. Boxed nucleotides are as in (A).
Figure 4
Figure 4
The 5' ends ofD. willistoni(Dwi) elements function as ribozymes. (A) RNA sequences from the 5' end of R2Dwi folded into the secondary structure previously determined for the ribozymes encoded by other Drosophila R2 (left). J = nucleotides joining paired regions; L = loop; P = base paired region [25]. Similar structures are presented for R2Dwi_SIDE (‘Short Internally Deleted Element’) (center) and R2/R1Dwi_SIDE (right) with nucleotide differences relative to the R2 element circled in blue. J1/2 sequences for each element type are presented below with nucleotide differences relative to R2Dwi boxed in blue. Nucleotides boxed in pink correspond to a stop codon found in most Drosophila R2 elements. Nucleotide circled in pink corresponds to a ‘U’ residue conserved in Drosophila R2 elements and the R2 SIDE but not the hybrid SIDEs. (B) A 5% polyacrylamide denaturing gel showing the in vitro generated RNAs from 5' junction templates starting 95 bp upstream of the R2 site (lanes 1 and 2) or 74 bp upstream of the R1 site (lane 3) and extending 5 to 10 bp downstream of the ribozyme structure. Lane numbers correspond to ribozyme structure in (A). The uncleaved RNA (solid circle) and self-cleaved products (open circles) are indicated for each ribozyme. The fraction of synthesized RNA undergoing self-cleavage (fc) is under each lane. Lane M, RNA length markers with sizes indicated.
Figure 5
Figure 5
R2/R1 hybrid SIDEs. (A) R1 insertions in the 28S gene in Drosophila outside the melanogaster group are flanked by a 14 bp target site duplication (arrows, upper diagram). In four species (bottom diagrams), a family of insertion elements bearing R2 ribozyme sequences (blue box) upstream of sequences with identity to R1 elements (orange box) was found in the R1 site flanked by the same 14 bp target site duplication. (B) The diagrams show the extent and level of sequence identity of each hybrid SIDE to the R1 and R2 elements in the same species. In the case of the R2/R1 SIDEs from Drosophila falleni, Drosophila innubila and Drosophila immigrans sequence identity to R1 was limited to six conserved segments found in all Drosophila R1 elements (red vertical lines; see Additional file 1). A portion of the sequence between the third and fourth conserved segments in R2/R1Dim_SIDE has 75% identity to ITS-1 of D. immigrans (green box). The lengths of the R2/R1 SIDEs are shown to the right.
Figure 6
Figure 6
The 5' ends ofDrosophila falleniandDrosophila innubilaelements function as ribozymes. (A) The RNA secondary structures and highlighted nucleotides for R2Dfa and R2/R1Dfa_SIDE are as described in Figure 4. The corresponding regions in the D. innubila elements are identical except for the boxed U in R2Dfa (A in R2Din) and the boxed G in the R2/R1Dfa_SIDE (A in R2/R1Din_SIDE). J1/2 sequences for the elements are shown below with nucleotide differences relative to R2Dfa boxed in blue. (B)In vitro cotranscription/cleavage assays as described in Figure 4.
Figure 7
Figure 7
The 5' ends ofDrosophila immigranselements function as ribozymes. (A) Folded RNA secondary structures, J1/2 sequencers, and highlighted nucleotides for R2Dim and R2/R1Dim_SIDE are as described in Figure 4. (B)In vitro cotranscription/cleavage assay as described in Figure 4.
Figure 8
Figure 8
In vivotemplate jump to the small nuclear RNA, snU12. (A) 5' R2 junction products from PCR amplification in Drosophila ambigua separated on a native, 8% polyacrylamide gel. Lane M, DNA length markers. (B) Diagrams of sequenced PCR products: 28S sequences (gray boxes); R2 sequences (blue boxes); snU12 sequences, yellow boxes. Long PCR products (12 clones) had a 48 bp deletion of upstream 28S sequences, 156 bp with sequence identity to the 5' end of snU12, and a 6 bp repeat at the snU12/R2 junction (arrowheads). Short products (eight clones) had typical 5' junctions that differed by zero to two non-templated nucleotides. (C)In vitro cotranscription/cleavage assay of RNA containing R2 sequences with the snU12 extension indicated self-cleavage only immediately upstream of the R2 sequences (lane 1, open circles). RNA constructs (see (D)) designed to promote self-cleavage upstream of the snU12 sequences did not self-cleave (lanes 2 and 3, solid circles). (D) Secondary structures of R2 with U12 extension (1) and two modified constructs. The substitution of two C’s in the P1 stem (2) and deletion of the 5' end of R2 (3) are highlighted in gray. Structure number corresponds to lane number in (C). Nomenclature and highlighted nucleotides are as described in Figure 4.
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