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Functional roles of 3'-terminal structures of template RNA during in vivo retrotransposition of non-LTR retrotransposon, R1Bm.

Anzai T, Osanai M, Hamada M, Fujiwara H - Nucleic Acids Res. (2005)

Bottom Line: R1Bm is a non-LTR retrotransposon found specifically within 28S rRNA genes of the silkworm.When the downstream sequence of 28S rDNA target was added to the 3' end of R1 unit, reverse transcription started exactly from the 3' end of 3'UTR and retrotransposition efficiency increased.These results indicate that 3'-terminal structure of template RNA including read-through region interacts with its target rDNA sequences of R1Bm, which plays important roles in initial process of TPRT in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo Bioscience Building 501, Kashiwa, 277-8562, Japan.

ABSTRACT
R1Bm is a non-LTR retrotransposon found specifically within 28S rRNA genes of the silkworm. Different from other non-LTR retrotransposons encoding two open reading frames (ORFs), R1Bm structurally lacks a poly (A) tract at its 3' end. To study how R1Bm initiates reverse transcription from the poly (A)-less template RNA, we established an in vivo retrotransposition system using recombinant baculovirus, and characterized retrotransposition activities of R1Bm. Target-primed reverse transcription (TPRT) of R1Bm occurred from the cleavage site generated by endonuclease (EN). The 147 bp of 3'-untranslated region (3'UTR) was essential for efficient retrotransposition of R1Bm. Even using the complete R1Bm element, however, reverse transcription started from various sites of the template RNA mostly with 5'-UG-3' or 5'-UGU-3' at their 3' ends, which are presumably base-paired with 3' end of the EN-digested 28S rDNA target sequence, 5'-AGTAGATAGGGACA-3'. When the downstream sequence of 28S rDNA target was added to the 3' end of R1 unit, reverse transcription started exactly from the 3' end of 3'UTR and retrotransposition efficiency increased. These results indicate that 3'-terminal structure of template RNA including read-through region interacts with its target rDNA sequences of R1Bm, which plays important roles in initial process of TPRT in vivo.

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Schematic representation of initial process of TPRT in the R1 element. (Top) First, R1-EN cleaves the non-coding bottom strand of 28S rDNA in the A–C junction. Boxes represent the 14 bp of TSD. (Middle) Then, the target DNA is partially denatured, allowing the UGU on the RNA template to base-pair with the loose target DNA. The template RNA is indicated by a gray line. In this model, the read-through 28S rRNA sequence is base-paired with the DNA target in longer region. During this process, RT of R1Bm may recognize the 3′UTR and base-paired region (open arrow), and place the RNA template at the accurate position for initiation of reverse transcription. (Bottom) Next, reverse transcription starts from the position next to the UGU sequence of template RNA, using the 3′-OH of A residue as primer.
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fig6: Schematic representation of initial process of TPRT in the R1 element. (Top) First, R1-EN cleaves the non-coding bottom strand of 28S rDNA in the A–C junction. Boxes represent the 14 bp of TSD. (Middle) Then, the target DNA is partially denatured, allowing the UGU on the RNA template to base-pair with the loose target DNA. The template RNA is indicated by a gray line. In this model, the read-through 28S rRNA sequence is base-paired with the DNA target in longer region. During this process, RT of R1Bm may recognize the 3′UTR and base-paired region (open arrow), and place the RNA template at the accurate position for initiation of reverse transcription. (Bottom) Next, reverse transcription starts from the position next to the UGU sequence of template RNA, using the 3′-OH of A residue as primer.

Mentions: For 72 h.p.i., 38 out of 40 clones were integrated into the specific site of 28S rDNA with the TSD. There was a wide variety in the R1 sites adjoining the 5′ end of TSD, suggesting that reverse transcription initiated from various positions of template RNA. Some clones contained the downstream AcNPV vector region, implying reverse transcription from read-through transcripts with the non-R1 sequence. It is noteworthy that 24 of 40 clones for 72 h.p.i. and also 13 out of 16 clones for 48 h.p.i. had the identical sequence (TG or TGT, shown within parentheses in Figure 3B) at the 3′ end of the RNA template region (i.e. the start site of reverse transcription). The 5′-UG-3′ or 5′-UGU-3′ sequences are presumably base-paired with the bottom strand of TSD, 5′-AGTAGATAGGGACA-3′, suggesting the interaction between the template RNA and the target 28S rDNA sequence in the initial process of TPRT (see Figure 6). According to this hypothesis, the reverse transcription should start from the position next to the TGT or TG sequences. Among 40 clones, only two clones (Figure 3B; denoted as +) inserted into the sequence other than R1-specific integration site, which is 180 bp upstream of TSD.


Functional roles of 3'-terminal structures of template RNA during in vivo retrotransposition of non-LTR retrotransposon, R1Bm.

Anzai T, Osanai M, Hamada M, Fujiwara H - Nucleic Acids Res. (2005)

Schematic representation of initial process of TPRT in the R1 element. (Top) First, R1-EN cleaves the non-coding bottom strand of 28S rDNA in the A–C junction. Boxes represent the 14 bp of TSD. (Middle) Then, the target DNA is partially denatured, allowing the UGU on the RNA template to base-pair with the loose target DNA. The template RNA is indicated by a gray line. In this model, the read-through 28S rRNA sequence is base-paired with the DNA target in longer region. During this process, RT of R1Bm may recognize the 3′UTR and base-paired region (open arrow), and place the RNA template at the accurate position for initiation of reverse transcription. (Bottom) Next, reverse transcription starts from the position next to the UGU sequence of template RNA, using the 3′-OH of A residue as primer.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1074724&req=5

fig6: Schematic representation of initial process of TPRT in the R1 element. (Top) First, R1-EN cleaves the non-coding bottom strand of 28S rDNA in the A–C junction. Boxes represent the 14 bp of TSD. (Middle) Then, the target DNA is partially denatured, allowing the UGU on the RNA template to base-pair with the loose target DNA. The template RNA is indicated by a gray line. In this model, the read-through 28S rRNA sequence is base-paired with the DNA target in longer region. During this process, RT of R1Bm may recognize the 3′UTR and base-paired region (open arrow), and place the RNA template at the accurate position for initiation of reverse transcription. (Bottom) Next, reverse transcription starts from the position next to the UGU sequence of template RNA, using the 3′-OH of A residue as primer.
Mentions: For 72 h.p.i., 38 out of 40 clones were integrated into the specific site of 28S rDNA with the TSD. There was a wide variety in the R1 sites adjoining the 5′ end of TSD, suggesting that reverse transcription initiated from various positions of template RNA. Some clones contained the downstream AcNPV vector region, implying reverse transcription from read-through transcripts with the non-R1 sequence. It is noteworthy that 24 of 40 clones for 72 h.p.i. and also 13 out of 16 clones for 48 h.p.i. had the identical sequence (TG or TGT, shown within parentheses in Figure 3B) at the 3′ end of the RNA template region (i.e. the start site of reverse transcription). The 5′-UG-3′ or 5′-UGU-3′ sequences are presumably base-paired with the bottom strand of TSD, 5′-AGTAGATAGGGACA-3′, suggesting the interaction between the template RNA and the target 28S rDNA sequence in the initial process of TPRT (see Figure 6). According to this hypothesis, the reverse transcription should start from the position next to the TGT or TG sequences. Among 40 clones, only two clones (Figure 3B; denoted as +) inserted into the sequence other than R1-specific integration site, which is 180 bp upstream of TSD.

Bottom Line: R1Bm is a non-LTR retrotransposon found specifically within 28S rRNA genes of the silkworm.When the downstream sequence of 28S rDNA target was added to the 3' end of R1 unit, reverse transcription started exactly from the 3' end of 3'UTR and retrotransposition efficiency increased.These results indicate that 3'-terminal structure of template RNA including read-through region interacts with its target rDNA sequences of R1Bm, which plays important roles in initial process of TPRT in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo Bioscience Building 501, Kashiwa, 277-8562, Japan.

ABSTRACT
R1Bm is a non-LTR retrotransposon found specifically within 28S rRNA genes of the silkworm. Different from other non-LTR retrotransposons encoding two open reading frames (ORFs), R1Bm structurally lacks a poly (A) tract at its 3' end. To study how R1Bm initiates reverse transcription from the poly (A)-less template RNA, we established an in vivo retrotransposition system using recombinant baculovirus, and characterized retrotransposition activities of R1Bm. Target-primed reverse transcription (TPRT) of R1Bm occurred from the cleavage site generated by endonuclease (EN). The 147 bp of 3'-untranslated region (3'UTR) was essential for efficient retrotransposition of R1Bm. Even using the complete R1Bm element, however, reverse transcription started from various sites of the template RNA mostly with 5'-UG-3' or 5'-UGU-3' at their 3' ends, which are presumably base-paired with 3' end of the EN-digested 28S rDNA target sequence, 5'-AGTAGATAGGGACA-3'. When the downstream sequence of 28S rDNA target was added to the 3' end of R1 unit, reverse transcription started exactly from the 3' end of 3'UTR and retrotransposition efficiency increased. These results indicate that 3'-terminal structure of template RNA including read-through region interacts with its target rDNA sequences of R1Bm, which plays important roles in initial process of TPRT in vivo.

Show MeSH
Related in: MedlinePlus