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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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3′-junction analysis for retrotransposed R1 elements. (A) PCR amplification of the 3′ boundaries between the transposed R1 and the 28S rDNA gene. Sf9 genomic DNAs were extracted 12, 24, 48, and 72 h post-infection (h.p.i.) with AcNPV expressing wild-type R1, 2H209A (EN-deficient mutant) and 2D680A (RT-deficient mutant). The purified DNA was used as template for PCR amplification with a pair of primers, +4941 and 28S(+109) (Figure 2A). The PCR products were subjected to 2.5% agarose electrophoresis and stained with ethidium bromide. The molecular size marker is loaded alongside and each size in base pair (bp) was shown on the left of the picture. (B and C) 72 h.p.i. 3′ junction clones obtained with wild-type R1 (B) and endonuclease-deficient R1 (2H209A) (C). Shown at the top of each figure is a diagram of the 3′ end structure of the construct. Sequences derived from the R1Bm and the pAcGHLTB vectors are indicated by shaded and open boxes, respectively. The initiation sites for reverse transcription (left of the dotted vertical lines) are indicated by nucleotide numbers. The target DNA regions are shown on the right of the dotted vertical lines. Extra nucleotides at the junction that are not derived from either the 28S gene or the R1 construct are given between the two vertical lines (non-templated). 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 and the most major type is indicated by an asterisk. The TGT or TG sequences on the 3′ end of the R1 template that can base-pair with the target DNA are also indicated (Figure 6). +, insertions into the site 180 bp upstream of TSD observed for wild-type and endonuclease-deficient R1.
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fig3: 3′-junction analysis for retrotransposed R1 elements. (A) PCR amplification of the 3′ boundaries between the transposed R1 and the 28S rDNA gene. Sf9 genomic DNAs were extracted 12, 24, 48, and 72 h post-infection (h.p.i.) with AcNPV expressing wild-type R1, 2H209A (EN-deficient mutant) and 2D680A (RT-deficient mutant). The purified DNA was used as template for PCR amplification with a pair of primers, +4941 and 28S(+109) (Figure 2A). The PCR products were subjected to 2.5% agarose electrophoresis and stained with ethidium bromide. The molecular size marker is loaded alongside and each size in base pair (bp) was shown on the left of the picture. (B and C) 72 h.p.i. 3′ junction clones obtained with wild-type R1 (B) and endonuclease-deficient R1 (2H209A) (C). Shown at the top of each figure is a diagram of the 3′ end structure of the construct. Sequences derived from the R1Bm and the pAcGHLTB vectors are indicated by shaded and open boxes, respectively. The initiation sites for reverse transcription (left of the dotted vertical lines) are indicated by nucleotide numbers. The target DNA regions are shown on the right of the dotted vertical lines. Extra nucleotides at the junction that are not derived from either the 28S gene or the R1 construct are given between the two vertical lines (non-templated). 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 and the most major type is indicated by an asterisk. The TGT or TG sequences on the 3′ end of the R1 template that can base-pair with the target DNA are also indicated (Figure 6). +, insertions into the site 180 bp upstream of TSD observed for wild-type and endonuclease-deficient R1.

Mentions: First, the 3′ junctions between the retrotransposed R1 element and the 28S gene were analyzed (Figure 3). A single round of PCR gave rise to the bands indicating the retrotransposition events at 48 and 72 h.p.i. of recombinant AcNPV constructs (Figure 3A). As seen in lane 4, there were only two bands of 400 and 600 bp at 48 h.p.i. At 72 h.p.i., the total intensity and the number of bands increased to the range of 200–1000 bp as in lane 5. In order to know whether these bands represent retrotransposed copies, we have cloned total PCR products into a cloning vector, and sequenced 16 clones for lane 4 (data no shown) and 40 clones for lane 5 (Figure 3B).


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)

3′-junction analysis for retrotransposed R1 elements. (A) PCR amplification of the 3′ boundaries between the transposed R1 and the 28S rDNA gene. Sf9 genomic DNAs were extracted 12, 24, 48, and 72 h post-infection (h.p.i.) with AcNPV expressing wild-type R1, 2H209A (EN-deficient mutant) and 2D680A (RT-deficient mutant). The purified DNA was used as template for PCR amplification with a pair of primers, +4941 and 28S(+109) (Figure 2A). The PCR products were subjected to 2.5% agarose electrophoresis and stained with ethidium bromide. The molecular size marker is loaded alongside and each size in base pair (bp) was shown on the left of the picture. (B and C) 72 h.p.i. 3′ junction clones obtained with wild-type R1 (B) and endonuclease-deficient R1 (2H209A) (C). Shown at the top of each figure is a diagram of the 3′ end structure of the construct. Sequences derived from the R1Bm and the pAcGHLTB vectors are indicated by shaded and open boxes, respectively. The initiation sites for reverse transcription (left of the dotted vertical lines) are indicated by nucleotide numbers. The target DNA regions are shown on the right of the dotted vertical lines. Extra nucleotides at the junction that are not derived from either the 28S gene or the R1 construct are given between the two vertical lines (non-templated). 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 and the most major type is indicated by an asterisk. The TGT or TG sequences on the 3′ end of the R1 template that can base-pair with the target DNA are also indicated (Figure 6). +, insertions into the site 180 bp upstream of TSD observed for wild-type and endonuclease-deficient R1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1074724&req=5

fig3: 3′-junction analysis for retrotransposed R1 elements. (A) PCR amplification of the 3′ boundaries between the transposed R1 and the 28S rDNA gene. Sf9 genomic DNAs were extracted 12, 24, 48, and 72 h post-infection (h.p.i.) with AcNPV expressing wild-type R1, 2H209A (EN-deficient mutant) and 2D680A (RT-deficient mutant). The purified DNA was used as template for PCR amplification with a pair of primers, +4941 and 28S(+109) (Figure 2A). The PCR products were subjected to 2.5% agarose electrophoresis and stained with ethidium bromide. The molecular size marker is loaded alongside and each size in base pair (bp) was shown on the left of the picture. (B and C) 72 h.p.i. 3′ junction clones obtained with wild-type R1 (B) and endonuclease-deficient R1 (2H209A) (C). Shown at the top of each figure is a diagram of the 3′ end structure of the construct. Sequences derived from the R1Bm and the pAcGHLTB vectors are indicated by shaded and open boxes, respectively. The initiation sites for reverse transcription (left of the dotted vertical lines) are indicated by nucleotide numbers. The target DNA regions are shown on the right of the dotted vertical lines. Extra nucleotides at the junction that are not derived from either the 28S gene or the R1 construct are given between the two vertical lines (non-templated). 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 and the most major type is indicated by an asterisk. The TGT or TG sequences on the 3′ end of the R1 template that can base-pair with the target DNA are also indicated (Figure 6). +, insertions into the site 180 bp upstream of TSD observed for wild-type and endonuclease-deficient R1.
Mentions: First, the 3′ junctions between the retrotransposed R1 element and the 28S gene were analyzed (Figure 3). A single round of PCR gave rise to the bands indicating the retrotransposition events at 48 and 72 h.p.i. of recombinant AcNPV constructs (Figure 3A). As seen in lane 4, there were only two bands of 400 and 600 bp at 48 h.p.i. At 72 h.p.i., the total intensity and the number of bands increased to the range of 200–1000 bp as in lane 5. In order to know whether these bands represent retrotransposed copies, we have cloned total PCR products into a cloning vector, and sequenced 16 clones for lane 4 (data no shown) and 40 clones for lane 5 (Figure 3B).

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