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Independently derived targeting of 28S rDNA by A- and D-clade R2 retrotransposons: Plasticity of integration mechanism.

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.

SUBMITTER: Thompson BK

PROVIDER: S-EPMC3190273 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Independently derived targeting of 28S rDNA by A- and D-clade R2 retrotransposons: Plasticity of integration mechanism.

Thompson Blaine K BK Christensen Shawn M SM

Mobile genetic elements 20110501 1

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. R ...[more]

PMID: 22016843

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Project description: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.

Project description:To facilitate the process of spiral nematode species delineation, populations of Helicotylenchus canadensis, H. pseudorobustus, and H. varicaudatus deriving from various locations and diverse natural and anthropogenic environments from Poland were investigated and characterized. For the first time, 28S rDNA sequences are reported for H. canadensis and H. varicaudatus, whereas new mtCOI sequences were acquired for all three analyzed species. A Bayesian phylogenetic analysis of the 28S rDNA fragments revealed that H. canadensis and H. varicaudatus are members of a clade that is a sister group to all other Helicotylenchus species; however, the closest known sister group to H. canadensis is H. vulgaris type A. Both 28S rDNA- and mtCOI-based phylogenetic results suggest that this clade excludes H. pseudorobustus, whose most recent common ancestor with the former species was inferred to be the ancestor of all Helicotylenchus species. Moreover, within the mtCOI sequences obtained from H. pseudorobustus, unlike from the other two, a simultaneous presence of TAG and TAA codons was identified. This may indicate mitochondrial genetic code alterations or other genomic rearrangements in H. pseudorobustus.To facilitate the process of spiral nematode species delineation, populations of Helicotylenchus canadensis, H. pseudorobustus, and H. varicaudatus deriving from various locations and diverse natural and anthropogenic environments from Poland were investigated and characterized. For the first time, 28S rDNA sequences are reported for H. canadensis and H. varicaudatus, whereas new mtCOI sequences were acquired for all three analyzed species. A Bayesian phylogenetic analysis of the 28S rDNA fragments revealed that H. canadensis and H. varicaudatus are members of a clade that is a sister group to all other Helicotylenchus species; however, the closest known sister group to H. canadensis is H. vulgaris type A. Both 28S rDNA- and mtCOI-based phylogenetic results suggest that this clade excludes H. pseudorobustus, whose most recent common ancestor with the former species was inferred to be the ancestor of all Helicotylenchus species. Moreover, within the mtCOI sequences obtained from H. pseudorobustus, unlike from the other two, a simultaneous presence of TAG and TAA codons was identified. This may indicate mitochondrial genetic code alterations or other genomic rearrangements in H. pseudorobustus.

Dataset Information

Independently derived targeting of 28S rDNA by A- and D-clade R2 retrotransposons: Plasticity of integration mechanism.

Publications

Independently derived targeting of 28S rDNA by A- and D-clade R2 retrotransposons: Plasticity of integration mechanism.

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