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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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Identification of a new subtype of the R1 element from the silkworm, Bombyx mori. (A) Schematic representation of 28S gene-specific LINEs. Shown in the middle is a diagram of the rDNA unit of Bombyx mori. The newly identified R1 clone (named R1Bmks) is integrated into the sequence 74 bp downstream of the R2 insertion site. Open reading frames (ORFs) are depicted by open boxes. The positions of non-synonymous mutations in the ORF1 and nucleotide insertions in the ORF2 (see B and C) are shown by white and black arrowheads, respectively. (B and C) Altered amino acid sequence of R1Bmks in the ORF2. Insertion of 3 nt within the RT domain results in alteration of 10 amino acids (indicated by bold line), compared with R1Bm (B). Addition of a single nucleotide in R1Bmks alters the amino acid sequences and made the length of ORF2 shorter by 12 amino acids, compared with R1Bm (C). The amino acids conserved between more than two elements are indicated by bold letters. Putative translational stop was indicated by asterisks. R1Bm clones are aligned with R1Dm (X51968; accession number) from D.melanogaster; R1Sc (L00945) from Sciara coprophila.
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fig1: Identification of a new subtype of the R1 element from the silkworm, Bombyx mori. (A) Schematic representation of 28S gene-specific LINEs. Shown in the middle is a diagram of the rDNA unit of Bombyx mori. The newly identified R1 clone (named R1Bmks) is integrated into the sequence 74 bp downstream of the R2 insertion site. Open reading frames (ORFs) are depicted by open boxes. The positions of non-synonymous mutations in the ORF1 and nucleotide insertions in the ORF2 (see B and C) are shown by white and black arrowheads, respectively. (B and C) Altered amino acid sequence of R1Bmks in the ORF2. Insertion of 3 nt within the RT domain results in alteration of 10 amino acids (indicated by bold line), compared with R1Bm (B). Addition of a single nucleotide in R1Bmks alters the amino acid sequences and made the length of ORF2 shorter by 12 amino acids, compared with R1Bm (C). The amino acids conserved between more than two elements are indicated by bold letters. Putative translational stop was indicated by asterisks. R1Bm clones are aligned with R1Dm (X51968; accession number) from D.melanogaster; R1Sc (L00945) from Sciara coprophila.

Mentions: R1Bm is a member of recently-branched subtype, and inserted specifically into the 28S rDNA of the silkworm (9,21). The R1 insertion site is located 74 bp downstream of the R2 site (Figure 1A) (22). R1Bm is flanked by specific 14 bp target site duplication (TSD), and lacks the poly (A) tract at its 3′ end. To understand the basal mechanisms of TPRT, it is of great interest to know how this poly (A)-less R1 initiates its reverse transcription. In this study, we have newly cloned the R1 element from the silkworm genome, and firstly succeeded in retrotransposing the R1 sequence into the 28S rDNA target of the host cells with recombinant baculovirus, AcNPV. Using the in vivo retrotransposition system of R1, we studied the functional roles of 3′UTR of R1 RNA and co-expressed 28S rRNA downstream of R1 sequence.


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)

Identification of a new subtype of the R1 element from the silkworm, Bombyx mori. (A) Schematic representation of 28S gene-specific LINEs. Shown in the middle is a diagram of the rDNA unit of Bombyx mori. The newly identified R1 clone (named R1Bmks) is integrated into the sequence 74 bp downstream of the R2 insertion site. Open reading frames (ORFs) are depicted by open boxes. The positions of non-synonymous mutations in the ORF1 and nucleotide insertions in the ORF2 (see B and C) are shown by white and black arrowheads, respectively. (B and C) Altered amino acid sequence of R1Bmks in the ORF2. Insertion of 3 nt within the RT domain results in alteration of 10 amino acids (indicated by bold line), compared with R1Bm (B). Addition of a single nucleotide in R1Bmks alters the amino acid sequences and made the length of ORF2 shorter by 12 amino acids, compared with R1Bm (C). The amino acids conserved between more than two elements are indicated by bold letters. Putative translational stop was indicated by asterisks. R1Bm clones are aligned with R1Dm (X51968; accession number) from D.melanogaster; R1Sc (L00945) from Sciara coprophila.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1074724&req=5

fig1: Identification of a new subtype of the R1 element from the silkworm, Bombyx mori. (A) Schematic representation of 28S gene-specific LINEs. Shown in the middle is a diagram of the rDNA unit of Bombyx mori. The newly identified R1 clone (named R1Bmks) is integrated into the sequence 74 bp downstream of the R2 insertion site. Open reading frames (ORFs) are depicted by open boxes. The positions of non-synonymous mutations in the ORF1 and nucleotide insertions in the ORF2 (see B and C) are shown by white and black arrowheads, respectively. (B and C) Altered amino acid sequence of R1Bmks in the ORF2. Insertion of 3 nt within the RT domain results in alteration of 10 amino acids (indicated by bold line), compared with R1Bm (B). Addition of a single nucleotide in R1Bmks alters the amino acid sequences and made the length of ORF2 shorter by 12 amino acids, compared with R1Bm (C). The amino acids conserved between more than two elements are indicated by bold letters. Putative translational stop was indicated by asterisks. R1Bm clones are aligned with R1Dm (X51968; accession number) from D.melanogaster; R1Sc (L00945) from Sciara coprophila.
Mentions: R1Bm is a member of recently-branched subtype, and inserted specifically into the 28S rDNA of the silkworm (9,21). The R1 insertion site is located 74 bp downstream of the R2 site (Figure 1A) (22). R1Bm is flanked by specific 14 bp target site duplication (TSD), and lacks the poly (A) tract at its 3′ end. To understand the basal mechanisms of TPRT, it is of great interest to know how this poly (A)-less R1 initiates its reverse transcription. In this study, we have newly cloned the R1 element from the silkworm genome, and firstly succeeded in retrotransposing the R1 sequence into the 28S rDNA target of the host cells with recombinant baculovirus, AcNPV. Using the in vivo retrotransposition system of R1, we studied the functional roles of 3′UTR of R1 RNA and co-expressed 28S rRNA downstream of R1 sequence.

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