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Control of chicken CR1 retrotransposons is independent of Dicer-mediated RNA interference pathway.

Lee SH, Eldi P, Cho SY, Rangasamy D - BMC Biol. (2009)

Bottom Line: Here, we describe how the loss of Dicer in chicken cells does not reactivate endogenous chicken CR1 retrotransposons with impaired RNAi machinery, suggesting that the control of chicken CR1 is independent of Dicer-induced RNAi silencing.In contrast, upon introduction of a functionally active human L1 retrotransposable element that contains an active 5' UTR promoter, the Dicer-deficient chicken cells show a strong increase in the accumulation of human L1 transcripts and retrotransposition activity, highlighting a major difference between chicken CR1 and other mammalian L1 retrotransposons.Our data provide evidence that chicken CR1 retrotransposons, unlike their mammalian L1 counterparts, do not undergo retrotransposition because most CR1 retrotransposons are truncated or mutated at their 5'UTR promoters and thus are not subjected to Dicer-mediated RNAi-silencing control.

View Article: PubMed Central - HTML - PubMed

Affiliation: The John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia. Sunghun.Lee@anu.edu.au

ABSTRACT

Background: Dicer is an RNase III-ribonuclease that initiates the formation of small interfering RNAs as a defence against genomic parasites such as retrotransposons. Despite intensive characterization in mammalian species, the biological functions of Dicer in controlling retrotransposable elements of the non-mammalian vertebrate are poorly understood. In this report, we examine the role of chicken Dicer in controlling the activity of chicken CR1 retrotransposable elements in a chicken-human hybrid DT40 cell line employing a conditional loss-of-Dicer function.

Results: Retrotransposition is detrimental to host genome stability and thus eukaryotic cells have developed mechanisms to limit the expansion of retrotransposons by Dicer-mediated RNAi silencing pathways. However, the mechanisms that control the activity and copy numbers of transposable elements in chicken remain unclear. Here, we describe how the loss of Dicer in chicken cells does not reactivate endogenous chicken CR1 retrotransposons with impaired RNAi machinery, suggesting that the control of chicken CR1 is independent of Dicer-induced RNAi silencing. In contrast, upon introduction of a functionally active human L1 retrotransposable element that contains an active 5' UTR promoter, the Dicer-deficient chicken cells show a strong increase in the accumulation of human L1 transcripts and retrotransposition activity, highlighting a major difference between chicken CR1 and other mammalian L1 retrotransposons.

Conclusion: Our data provide evidence that chicken CR1 retrotransposons, unlike their mammalian L1 counterparts, do not undergo retrotransposition because most CR1 retrotransposons are truncated or mutated at their 5'UTR promoters and thus are not subjected to Dicer-mediated RNAi-silencing control.

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Chicken DT40 cell culture-based assay for L1 retrotransposition. (A) Schematic of the human L1-eGFP vector. The L1 transcription is driven by a CMV promoter in addition to the L1 5'UTR. The human L1 retrotransposon contains an intron-interrupted EGFP reporter in the 3'UTR region with its own promoter and polyadenylation signal. The EGFP cassette is in antisense orientation relative to L1. Only when EGFP is transcribed from the L1 promoter, spliced, reverse-transcribed and integrated into the genome does a cell become GFP-positive. As a negative control, inactive L1 (pCMV-ΔRP99-eGFP) that contained two missense mutations in ORF1 that abolish retrotransposition was used. Arrows depict the location of the geno-5 (left) and geno-3 (right) primers used in the PCR assay shown in below. SD = splice donor; SA = splice acceptor. (B) Detection of L1 retrotransposition events in chicken cells. The geno-5 and geno-3 primers that flank the intron in GFP were used for PCR and analysed on a 1.2% agarose gel. PCR products of 1.49 kb (corresponding to the intron-containing transgene) and approximately 0.5 kb that lacks the 909 bp intron (corresponding to the transposed insertion) are shown. Negative, genomic DNA from wild-type DT40 cells; Vector, 5 ng plasmid DNA; Marker, 1 kb-plus DNA marker (Invitogen). (C) Quantitative RT-PCR analysis of the L1 transcript of human L1 elements in control (Dox-) and Dicer-deficient (Dox+) cells after addition of 2 μg/ml Dox for 72 h. Data are normalized to that of chicken β-actin transcripts. Error bars show s.d. (n = 6).
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Figure 2: Chicken DT40 cell culture-based assay for L1 retrotransposition. (A) Schematic of the human L1-eGFP vector. The L1 transcription is driven by a CMV promoter in addition to the L1 5'UTR. The human L1 retrotransposon contains an intron-interrupted EGFP reporter in the 3'UTR region with its own promoter and polyadenylation signal. The EGFP cassette is in antisense orientation relative to L1. Only when EGFP is transcribed from the L1 promoter, spliced, reverse-transcribed and integrated into the genome does a cell become GFP-positive. As a negative control, inactive L1 (pCMV-ΔRP99-eGFP) that contained two missense mutations in ORF1 that abolish retrotransposition was used. Arrows depict the location of the geno-5 (left) and geno-3 (right) primers used in the PCR assay shown in below. SD = splice donor; SA = splice acceptor. (B) Detection of L1 retrotransposition events in chicken cells. The geno-5 and geno-3 primers that flank the intron in GFP were used for PCR and analysed on a 1.2% agarose gel. PCR products of 1.49 kb (corresponding to the intron-containing transgene) and approximately 0.5 kb that lacks the 909 bp intron (corresponding to the transposed insertion) are shown. Negative, genomic DNA from wild-type DT40 cells; Vector, 5 ng plasmid DNA; Marker, 1 kb-plus DNA marker (Invitogen). (C) Quantitative RT-PCR analysis of the L1 transcript of human L1 elements in control (Dox-) and Dicer-deficient (Dox+) cells after addition of 2 μg/ml Dox for 72 h. Data are normalized to that of chicken β-actin transcripts. Error bars show s.d. (n = 6).

Mentions: To further confirm that the Dicer is indeed required for controlling human L1 expression and retrotransposition, we introduced L1 expression cassettes harbouring a retrotransposition indicator for cell culture-based assay [11,19]. This cassette consists of a full-length human L1 tagged at its 3'UTR with an antisense enhanced green fluorescent protein (EGFP) gene, which is driven by a cytomegalovirus (CMV) promoter (Figure 2A). The EGFP gene is disrupted by a γ-globin intron in the same orientation as the L1 transcript. This arrangement ensures that EGFP expression occurs only after L1 transcription, splicing of the intron, reverse-transcription, and insertion of the L1 copy back into chromosomal DNA (that is, after the retrotransposition event).


Control of chicken CR1 retrotransposons is independent of Dicer-mediated RNA interference pathway.

Lee SH, Eldi P, Cho SY, Rangasamy D - BMC Biol. (2009)

Chicken DT40 cell culture-based assay for L1 retrotransposition. (A) Schematic of the human L1-eGFP vector. The L1 transcription is driven by a CMV promoter in addition to the L1 5'UTR. The human L1 retrotransposon contains an intron-interrupted EGFP reporter in the 3'UTR region with its own promoter and polyadenylation signal. The EGFP cassette is in antisense orientation relative to L1. Only when EGFP is transcribed from the L1 promoter, spliced, reverse-transcribed and integrated into the genome does a cell become GFP-positive. As a negative control, inactive L1 (pCMV-ΔRP99-eGFP) that contained two missense mutations in ORF1 that abolish retrotransposition was used. Arrows depict the location of the geno-5 (left) and geno-3 (right) primers used in the PCR assay shown in below. SD = splice donor; SA = splice acceptor. (B) Detection of L1 retrotransposition events in chicken cells. The geno-5 and geno-3 primers that flank the intron in GFP were used for PCR and analysed on a 1.2% agarose gel. PCR products of 1.49 kb (corresponding to the intron-containing transgene) and approximately 0.5 kb that lacks the 909 bp intron (corresponding to the transposed insertion) are shown. Negative, genomic DNA from wild-type DT40 cells; Vector, 5 ng plasmid DNA; Marker, 1 kb-plus DNA marker (Invitogen). (C) Quantitative RT-PCR analysis of the L1 transcript of human L1 elements in control (Dox-) and Dicer-deficient (Dox+) cells after addition of 2 μg/ml Dox for 72 h. Data are normalized to that of chicken β-actin transcripts. Error bars show s.d. (n = 6).
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Figure 2: Chicken DT40 cell culture-based assay for L1 retrotransposition. (A) Schematic of the human L1-eGFP vector. The L1 transcription is driven by a CMV promoter in addition to the L1 5'UTR. The human L1 retrotransposon contains an intron-interrupted EGFP reporter in the 3'UTR region with its own promoter and polyadenylation signal. The EGFP cassette is in antisense orientation relative to L1. Only when EGFP is transcribed from the L1 promoter, spliced, reverse-transcribed and integrated into the genome does a cell become GFP-positive. As a negative control, inactive L1 (pCMV-ΔRP99-eGFP) that contained two missense mutations in ORF1 that abolish retrotransposition was used. Arrows depict the location of the geno-5 (left) and geno-3 (right) primers used in the PCR assay shown in below. SD = splice donor; SA = splice acceptor. (B) Detection of L1 retrotransposition events in chicken cells. The geno-5 and geno-3 primers that flank the intron in GFP were used for PCR and analysed on a 1.2% agarose gel. PCR products of 1.49 kb (corresponding to the intron-containing transgene) and approximately 0.5 kb that lacks the 909 bp intron (corresponding to the transposed insertion) are shown. Negative, genomic DNA from wild-type DT40 cells; Vector, 5 ng plasmid DNA; Marker, 1 kb-plus DNA marker (Invitogen). (C) Quantitative RT-PCR analysis of the L1 transcript of human L1 elements in control (Dox-) and Dicer-deficient (Dox+) cells after addition of 2 μg/ml Dox for 72 h. Data are normalized to that of chicken β-actin transcripts. Error bars show s.d. (n = 6).
Mentions: To further confirm that the Dicer is indeed required for controlling human L1 expression and retrotransposition, we introduced L1 expression cassettes harbouring a retrotransposition indicator for cell culture-based assay [11,19]. This cassette consists of a full-length human L1 tagged at its 3'UTR with an antisense enhanced green fluorescent protein (EGFP) gene, which is driven by a cytomegalovirus (CMV) promoter (Figure 2A). The EGFP gene is disrupted by a γ-globin intron in the same orientation as the L1 transcript. This arrangement ensures that EGFP expression occurs only after L1 transcription, splicing of the intron, reverse-transcription, and insertion of the L1 copy back into chromosomal DNA (that is, after the retrotransposition event).

Bottom Line: Here, we describe how the loss of Dicer in chicken cells does not reactivate endogenous chicken CR1 retrotransposons with impaired RNAi machinery, suggesting that the control of chicken CR1 is independent of Dicer-induced RNAi silencing.In contrast, upon introduction of a functionally active human L1 retrotransposable element that contains an active 5' UTR promoter, the Dicer-deficient chicken cells show a strong increase in the accumulation of human L1 transcripts and retrotransposition activity, highlighting a major difference between chicken CR1 and other mammalian L1 retrotransposons.Our data provide evidence that chicken CR1 retrotransposons, unlike their mammalian L1 counterparts, do not undergo retrotransposition because most CR1 retrotransposons are truncated or mutated at their 5'UTR promoters and thus are not subjected to Dicer-mediated RNAi-silencing control.

View Article: PubMed Central - HTML - PubMed

Affiliation: The John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia. Sunghun.Lee@anu.edu.au

ABSTRACT

Background: Dicer is an RNase III-ribonuclease that initiates the formation of small interfering RNAs as a defence against genomic parasites such as retrotransposons. Despite intensive characterization in mammalian species, the biological functions of Dicer in controlling retrotransposable elements of the non-mammalian vertebrate are poorly understood. In this report, we examine the role of chicken Dicer in controlling the activity of chicken CR1 retrotransposable elements in a chicken-human hybrid DT40 cell line employing a conditional loss-of-Dicer function.

Results: Retrotransposition is detrimental to host genome stability and thus eukaryotic cells have developed mechanisms to limit the expansion of retrotransposons by Dicer-mediated RNAi silencing pathways. However, the mechanisms that control the activity and copy numbers of transposable elements in chicken remain unclear. Here, we describe how the loss of Dicer in chicken cells does not reactivate endogenous chicken CR1 retrotransposons with impaired RNAi machinery, suggesting that the control of chicken CR1 is independent of Dicer-induced RNAi silencing. In contrast, upon introduction of a functionally active human L1 retrotransposable element that contains an active 5' UTR promoter, the Dicer-deficient chicken cells show a strong increase in the accumulation of human L1 transcripts and retrotransposition activity, highlighting a major difference between chicken CR1 and other mammalian L1 retrotransposons.

Conclusion: Our data provide evidence that chicken CR1 retrotransposons, unlike their mammalian L1 counterparts, do not undergo retrotransposition because most CR1 retrotransposons are truncated or mutated at their 5'UTR promoters and thus are not subjected to Dicer-mediated RNAi-silencing control.

Show MeSH