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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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Effects of downstream sequences on the R1 retrotransposition. (A) Diagram of the R1 constructs with various 3′ end structures; 14 and 50 nt of the downstream 28S gene are added to the R1WT construct. The 14 bp sequence of TSD is indicated by an open box. Note that the 3′ termini of each construct is followed by the AcNPV-derived polyhedrin 3′UTR. Transcription start, +1. (B) PCR amplification of the 3′ boundaries between the transposed R1 copies and the 28S gene. DNA extraction and PCR reaction are basically same as in Figure 3. The primer set used for PCR was +5121 and 28S(+109). 2D680A, RT-deficient mutant. (C) 72 h.p.i. 3′ junctions obtained with various R1 construct in (A). Shown at the top of each figure is a diagram of the 3′ end structure of the constructs. Sequences derived from R1Bm and additional 28S downstream sequences are indicated by shaded and hatched boxes, respectively. The vertical dotted lines represent the boundary between the cDNA region and the target DNA region. The initiation site for reverse transcription (left of the dotted vertical lines) is indicated by the numbers. Boxes to the right of the vertical lines represent the 14 bp of TSD. The number of clones containing each insertion type is indicated in the right-most column. Two clones amplified from the + (A)5 + 14 nt construct contained mutations at the 3′ end of 3′UTR (G of GAA corresponds to the position +5446).
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fig5: Effects of downstream sequences on the R1 retrotransposition. (A) Diagram of the R1 constructs with various 3′ end structures; 14 and 50 nt of the downstream 28S gene are added to the R1WT construct. The 14 bp sequence of TSD is indicated by an open box. Note that the 3′ termini of each construct is followed by the AcNPV-derived polyhedrin 3′UTR. Transcription start, +1. (B) PCR amplification of the 3′ boundaries between the transposed R1 copies and the 28S gene. DNA extraction and PCR reaction are basically same as in Figure 3. The primer set used for PCR was +5121 and 28S(+109). 2D680A, RT-deficient mutant. (C) 72 h.p.i. 3′ junctions obtained with various R1 construct in (A). Shown at the top of each figure is a diagram of the 3′ end structure of the constructs. Sequences derived from R1Bm and additional 28S downstream sequences are indicated by shaded and hatched boxes, respectively. The vertical dotted lines represent the boundary between the cDNA region and the target DNA region. The initiation site for reverse transcription (left of the dotted vertical lines) is indicated by the numbers. Boxes to the right of the vertical lines represent the 14 bp of TSD. The number of clones containing each insertion type is indicated in the right-most column. Two clones amplified from the + (A)5 + 14 nt construct contained mutations at the 3′ end of 3′UTR (G of GAA corresponds to the position +5446).

Mentions: R2 element, 28S rDNA-specific LINE with a single ORF, requires several regions in 3′UTR for initiating the target-primed reverse transcription in vitro (11). To clarify the function of 3′UTR in the R1 retrotransposition, we made an R1 Δ3′UTR-AcNPV construct which lacks the entire R1 3′UTR (Figures 5A and C). Note that this construct retains a downstream polyhedrin 3′UTR. As shown in Figure 5B (Δ3′UTR; lane 4), the band was very faint, but two clones representing the authentic reverse transcription from ORF2 sequence of R1 were recovered from the PCR product (Figure 5C, Δ3′UTR). This result suggests that 3′UTR is not necessary absolutely for the R1 retrotransposition although loss of the sequence lowered severely the retrotransposition efficiency.


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)

Effects of downstream sequences on the R1 retrotransposition. (A) Diagram of the R1 constructs with various 3′ end structures; 14 and 50 nt of the downstream 28S gene are added to the R1WT construct. The 14 bp sequence of TSD is indicated by an open box. Note that the 3′ termini of each construct is followed by the AcNPV-derived polyhedrin 3′UTR. Transcription start, +1. (B) PCR amplification of the 3′ boundaries between the transposed R1 copies and the 28S gene. DNA extraction and PCR reaction are basically same as in Figure 3. The primer set used for PCR was +5121 and 28S(+109). 2D680A, RT-deficient mutant. (C) 72 h.p.i. 3′ junctions obtained with various R1 construct in (A). Shown at the top of each figure is a diagram of the 3′ end structure of the constructs. Sequences derived from R1Bm and additional 28S downstream sequences are indicated by shaded and hatched boxes, respectively. The vertical dotted lines represent the boundary between the cDNA region and the target DNA region. The initiation site for reverse transcription (left of the dotted vertical lines) is indicated by the numbers. Boxes to the right of the vertical lines represent the 14 bp of TSD. The number of clones containing each insertion type is indicated in the right-most column. Two clones amplified from the + (A)5 + 14 nt construct contained mutations at the 3′ end of 3′UTR (G of GAA corresponds to the position +5446).
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Related In: Results  -  Collection

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fig5: Effects of downstream sequences on the R1 retrotransposition. (A) Diagram of the R1 constructs with various 3′ end structures; 14 and 50 nt of the downstream 28S gene are added to the R1WT construct. The 14 bp sequence of TSD is indicated by an open box. Note that the 3′ termini of each construct is followed by the AcNPV-derived polyhedrin 3′UTR. Transcription start, +1. (B) PCR amplification of the 3′ boundaries between the transposed R1 copies and the 28S gene. DNA extraction and PCR reaction are basically same as in Figure 3. The primer set used for PCR was +5121 and 28S(+109). 2D680A, RT-deficient mutant. (C) 72 h.p.i. 3′ junctions obtained with various R1 construct in (A). Shown at the top of each figure is a diagram of the 3′ end structure of the constructs. Sequences derived from R1Bm and additional 28S downstream sequences are indicated by shaded and hatched boxes, respectively. The vertical dotted lines represent the boundary between the cDNA region and the target DNA region. The initiation site for reverse transcription (left of the dotted vertical lines) is indicated by the numbers. Boxes to the right of the vertical lines represent the 14 bp of TSD. The number of clones containing each insertion type is indicated in the right-most column. Two clones amplified from the + (A)5 + 14 nt construct contained mutations at the 3′ end of 3′UTR (G of GAA corresponds to the position +5446).
Mentions: R2 element, 28S rDNA-specific LINE with a single ORF, requires several regions in 3′UTR for initiating the target-primed reverse transcription in vitro (11). To clarify the function of 3′UTR in the R1 retrotransposition, we made an R1 Δ3′UTR-AcNPV construct which lacks the entire R1 3′UTR (Figures 5A and C). Note that this construct retains a downstream polyhedrin 3′UTR. As shown in Figure 5B (Δ3′UTR; lane 4), the band was very faint, but two clones representing the authentic reverse transcription from ORF2 sequence of R1 were recovered from the PCR product (Figure 5C, Δ3′UTR). This result suggests that 3′UTR is not necessary absolutely for the R1 retrotransposition although loss of the sequence lowered severely the retrotransposition efficiency.

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