Downstream 28S Gene Sequences on the RNA Template Affect the Choice of Primer and the Accuracy of Initiation by the R2 Reverse Transcriptase

Abstract

R2 non-long terminal repeat retrotransposable elements insert at a unique site in the 28S rRNA genes of insects. The protein encoded by the single open reading frame of R2 is capable of conducting the initial steps of its integration in vitro. The protein nicks the noncoding strand of the 28S target DNA (the strand which serves as a template for RNA synthesis) and uses the 3 hydroxyl group exposed by this nick to prime reverse transcription of the R2 RNA template. This target-primed reverse transcription (TPRT) reaction requires that the RNA template contains the 250-nucleotide 3 untranslated region of the R2 element. If this RNA template ends at the precise 3 end of the R2 element, then extra nucleotides, which we refer to as nontemplated nucleotides, are added to the target before cDNA synthesis. The presence of downstream 28S gene sequences on the RNA template reduces the total efficiency but eliminates these nontemplated additions, resulting in nearly 90% of all TPRT products reproducing the 3 junctions seen in vivo. Templates with 5 to 10 nucleotides of the 28S sequence are used most efficiently in this in vitro TPRT reaction. The requirement for downstream 28S rRNA sequences probably explains why the R2 elements of most insects differ from the majority of non-long terminal repeat retrotransposons in that they do not contain an A-rich repeat at their 3 junction with the target DNA. The presence of downstream sequences on these in vitro R2 templates also revealed that the R2 reverse transcriptase can prime cDNA synthesis by using the 3 end of another RNA molecule. This RNA-primed cDNA synthesis is not based on sequence complementarity between the RNA primer and the R2 template. The ability to use the 3 end of a noncomplementary RNA molecule has also been seen with the reverse transcriptase of the mitochondrial Mauriceville plasmid of Neurospora crassa.Non-long terminal repeat (non-LTR) retrotransposable elements are an abundant family of mobile elements found in most eukaryotes. The distinguishing features of these elements are that they encode an open reading frame with homology to reverse transcriptases but are not flanked by the terminal repeats associated with all retroviruses and all retrotransposable elements of the copia/Ty1 and gypsy/Ty3 groups (2,11,34). Evidence that the non-LTR retrotransposons encode a functional reverse transcriptase has been obtained for the jockey element of Drosophila melanogaster (18), CRE1 of Crithidia fasciculata (13), L1 of humans (26), and R2 of Bombyx mori (25). Direct evidence that these elements utilize an RNA intermediate in their mobility has been obtained from experiments in which inserted intron sequences were found to be accurately removed during the formation of new copies of the I element of D. melanogaster (28) and the L1 element of mice (12,20).Knowledge of the retrotransposition mechanism used by the non-LTR retrotransposable elements is still limited. Current models suggest that non-LTR elements retrotranspose by using the 3Ј-hydroxyl group at a DNA break t...

Secondary structure models of the 3′ untranslated regions of diverse R2 RNAs

“…7) serves a conserved role in each structure. The importance of this helix at the 3Ј end is consistent with deletion experiments showing that the 3Ј end of the sequence of B. mori is important for efficient TPRT (Luan and Eickbush 1995). A second invariably conserved feature of all four structures is a helix (helix A in Fig.…”

“…1A). The ability of the RNA to be positioned opposite the nicked site does not involve annealing of the RNA with the downstream DNA sequences (Luan and Eickbush 1996). It is not known whether RNA recognition and RNA positioning involve the same or different motifs.…”

“…Labeled cDNA products of variable lengths were generated because TPRT initiates at variable positions within such poly(A) tails, and the reverse transcriptase is able to add additional residues to the DNA target by slippage before firmly engaging the RNA (Luan and Eickbush 1996). For example, most of the R2 Dmer TPRT products resulted from initiation within the poly(A) tail, whereas the R2Fa products were highly variable in length with most containing longer tails, generated by slippage.…”

“…Each RNA template contains ∼250 nt from the 3Ј end of the R2 element as well as 20 nt from the flanking downstream 28S gene sequences. Reverse transcription is initiated at the junction between the R2 and 28S gene sequences, not at the 3Ј end of the RNA (Luan and Eickbush 1996;Bibillo and Eickbush 2002).…”

“…The R2 TPRT reaction in Bombyx mori requires the RNA sequence corresponding to the 250-nucleotide 3Ј untranslated region (3ЈUTR) of the R2 element (Luan et al 1993;Luan and Eickbush 1995). Previously, the R2-encoded protein from B. mori was also found to conduct TPRT by using RNA from the R2 element of Drosophila melanogaster (Mathews et al 1997).…”