R2 elements are non-long terminal repeat retrotransposons that specifically insert into 28S rRNA genes of many animal groups. These elements encode a single protein with reverse transcriptase and endonuclease activities as well as specific DNA and RNA binding properties. In this report, gel shift experiments were conducted to investigate the stoichiometry of the DNA, RNA, and protein components of the integration reaction. The enzymatic functions associated with each of the protein complexes were also determined, and DNase I digests were used to footprint the protein onto the target DNA. Additionally, a short polypeptide containing the N-terminal putative DNA-binding motifs was footprinted on the DNA target site. These combined findings revealed that one protein subunit binds the R2 RNA template and the DNA 10 to 40 bp upstream of the insertion site. This subunit cleaves the first DNA strand and uses that cleavage to prime reverse transcription of the R2 RNA transcript. Another protein subunit(s) uses the N-terminal DNA binding motifs to bind to the 18 bp of target DNA downstream of the insertion site and is responsible for cleavage of the second DNA strand. A complete model for the R2 integration reaction is presented, which with minor modifications is adaptable to other non-LTR retrotransposons.While originally viewed as the unique property of retroviruses, the reverse transcription of RNA templates is now known to be a mechanism used by many eukaryotic mobile elements. One class of elements, frequently referred to as the LTR retrotransposons because they contain long terminal repeats, utilize the same replication mechanism as retroviruses (reviewed in reference 33). Reverse transcription of the RNA template is usually primed by the 3Ј end of a tRNA annealed to the template. Full-length first and second DNA strands are made from the RNA template by the polymerase using the terminal repeats to jump from one end of the template to the other. The linear DNA product generated by reverse transcription is then inserted into chromosomal sites by an integrase.A second class of elements, usually referred to as the non-LTR retrotransposons because they lack terminal repeats, uses a different mechanism of integration. In this mechanism the chromosomal DNA target site is cleaved by an endonuclease, and the 3Ј end generated by this cleavage is used to prime the reverse transcription directly onto the DNA target (Fig. 1A). This target-primed reverse transcription, or TPRT mechanism, has been most comprehensively documented for the R2 element of Bombyx mori (22), but in vitro and in vivo assays involving other elements are consistent with the basic features of the TPRT model (7,11,25,32). One side effect of not requiring precise terminal repeats in any step of the reaction is that the reverse transcriptase of non-LTR retrotransposons is able to reverse transcribe other cellular RNA templates. Thus, the TPRT mechanism has been shown to generate short interspersed nuclear element (e.g., Alu) insertions as well as processed pse...

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5 end of the R2 element. In the absence of this 5 RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3 untranslated region of the R2 transcript is present. In the presence of the 320-nt 5 RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5 RNA is removed from this subunit. We postulate that the removal of the 5 RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first-and second-strand DNA cleavage in the R2 retrotransposition reaction.endonuclease ͉ retrotransposition ͉ reverse transcription ͉ RNA-protein interactions N on-LTR retrotransposons, also referred to as long interspersed nuclear elements (LINEs), are abundant insertions in many eukaryotic genomes. For example, there are Ͼ800,000 copies of these elements in the human genome, representing 17% of our DNA (1). Whereas retrotransposition assays in tissue culture cells have been developed to study non-LTR retrotransposition, many questions concerning the mechanism of their integration remain unanswered (2-5).R2 is a non-LTR retrotransposable element with rigid sequence specificity for a target site in the 28S rRNA genes of arthropods, platyhelminths, tunicates, and vertebrates (6, 7). The sequence specificity of R2 integration has enabled detailed biochemical studies of its retrotransposition reaction (Fig. 1A). We have previously shown that one R2 protein subunit of a probable dimer binds a 30-bp DNA segment upstream of the insertion site and cleaves the first strand (bottom strand, Fig. 1 A) of the target DNA (8, 9). If RNA corresponding to the 3Ј UTR of the R2 element is present, then this subunit primes reverse transcription of the R2 RNA transcript from the free 3Ј end released by the cleavage. This process is referred to as targetprimed reverse transcription (TPRT) (10). After reverse transcription, the second (top) DNA strand is cleaved by the second protein subunit, which binds a different DNA sequence downstream of the insertion site (9). We have postulated that this second R2 subunit is responsible for the synthesis of the second DNA strand and thereby completes the retrotransposition reaction (9).One ...

LNA (locked nucleic acids, i.e. oligonucleotides with a methyl bridge between the 2′ oxygen and 4′ carbon of ribose) and 2,6-diaminopurine were incorporated into 2′-O-methyl RNA pentamer and hexamer probes to make a microarray that binds unpaired RNA approximately isoenergetically. That is, binding is roughly independent of target sequence if target is unfolded. The isoenergetic binding and short probe length simplify interpretation of binding to a structured RNA to provide insight into target RNA secondary structure. Microarray binding and chemical mapping were used to probe the secondary structure of a 323 nt segment of the 5′ coding region of the R2 retrotransposon from Bombyx mori (R2Bm 5′ RNA). This R2Bm 5′ RNA orchestrates functioning of the R2 protein responsible for cleaving the second strand of DNA during insertion of the R2 sequence into the genome. The experimental results were used as constraints in a free energy minimization algorithm to provide an initial model for the secondary structure of the R2Bm 5′ RNA.

SummarySequences from the 5′ region of R2 retrotransposons of four species of silk moth are reported. In Bombyx mori, this region of the R2 messenger RNA contains a binding site for R2 protein and mediates interactions critical to R2 element insertion into the host genome. A model of secondary structure for this RNA fragment is proposed on the basis of binding to oligonucleotide microarrays, chemical mapping, and comparative sequence analysis. Five regions of conserved secondary structure are identified, including a novel pseudoknot. There is an apparent transition from an entirely RNA structure coding function in most of the 5′ segment of the fragment to a protein coding function in the 3′ segment. This suggests that regions evolved under separate functional constraints (structural, coding, or both).