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Search for limiting factors in the RNAi pathway in silkmoth tissues and the Bm5 cell line: the RNA-binding proteins R2D2 and Translin.

Swevers L, Liu J, Huvenne H, Smagghe G - PLoS ONE (2011)

Bottom Line: It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues.Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells.The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

View Article: PubMed Central - PubMed

Affiliation: Insect Molecular Genetics and Biotechnology, Institute of Biology, National Centre for Scientific Research Demokritos, Athens, Greece. swevers@bio.demokritos.gr

ABSTRACT
RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

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RNAi functional assays.A. Functional assay after transfection of dsRNA. Bm5 cells were transfected with DNA and either non-specific dsRNA or specific dsRNA at different ratios in the absence (Left) or presence (Right) of TcR2D2 expression plasmid. Indicated is the amount of luminescence in comparison with cells that received an equivalent amount of non-specific DNA (C; 100%). Experimental conditions include treatment with BmCAP-dsRNA and Luc-dsRNA at the indicated amounts (µg/ml in transfection mixture). N = 3. B. Functional assay after addition of nucleic acid (DNA or dsRNA) to the tissue culture medium. Cells were soaked with extracellular dsRNA for two days before transfection and for three days after transfection. Experimental conditions include treatment with DNA, BmCAP-dsRNA, BmHR3-dsRNA, Luc-dsRNA and GFP-dsRNA at the indicated concentrations (in µg/ml). Left: Luciferase assays to evaluate expression of the constitutive A-Luc transgene. Indicated is the luminescence/fluorescence ratio (N = 3). Right. Relative fluorescence measurements (fluorescence/luminescence ratios) 24 hrs after induction of the ecdysone ERE-gfp reporter with 200 nM of ecdysone agonist. Cells were soaked with nucleic acid (DNA or dsRNA) for two days before transfection and for another three days after transfection. At two days after transfection, cells were induced with hormone agonist for a period of 24 hours (N = 3).
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pone-0020250-g003: RNAi functional assays.A. Functional assay after transfection of dsRNA. Bm5 cells were transfected with DNA and either non-specific dsRNA or specific dsRNA at different ratios in the absence (Left) or presence (Right) of TcR2D2 expression plasmid. Indicated is the amount of luminescence in comparison with cells that received an equivalent amount of non-specific DNA (C; 100%). Experimental conditions include treatment with BmCAP-dsRNA and Luc-dsRNA at the indicated amounts (µg/ml in transfection mixture). N = 3. B. Functional assay after addition of nucleic acid (DNA or dsRNA) to the tissue culture medium. Cells were soaked with extracellular dsRNA for two days before transfection and for three days after transfection. Experimental conditions include treatment with DNA, BmCAP-dsRNA, BmHR3-dsRNA, Luc-dsRNA and GFP-dsRNA at the indicated concentrations (in µg/ml). Left: Luciferase assays to evaluate expression of the constitutive A-Luc transgene. Indicated is the luminescence/fluorescence ratio (N = 3). Right. Relative fluorescence measurements (fluorescence/luminescence ratios) 24 hrs after induction of the ecdysone ERE-gfp reporter with 200 nM of ecdysone agonist. Cells were soaked with nucleic acid (DNA or dsRNA) for two days before transfection and for another three days after transfection. At two days after transfection, cells were induced with hormone agonist for a period of 24 hours (N = 3).

Mentions: It was observed that intracellular RNAi occurs efficiently in Bm5 cells in the absence of BmR2D2 expression. Co-transfection of the actin-luciferase reporter with Luc-dsRNA results in a dose-dependent reduction of luciferase activity (Figure 3A, left panel). This response is specific, since a high dose of a non-specific dsRNA (BmCAP-dsRNA) did not result in a decrease in luciferase activity. On the other hand, soaking of Bm5 cells with dsRNA in the extracellular medium at high concentration did not result in gene silencing (Figure 3B, right panel) as was observed before [22].


Search for limiting factors in the RNAi pathway in silkmoth tissues and the Bm5 cell line: the RNA-binding proteins R2D2 and Translin.

Swevers L, Liu J, Huvenne H, Smagghe G - PLoS ONE (2011)

RNAi functional assays.A. Functional assay after transfection of dsRNA. Bm5 cells were transfected with DNA and either non-specific dsRNA or specific dsRNA at different ratios in the absence (Left) or presence (Right) of TcR2D2 expression plasmid. Indicated is the amount of luminescence in comparison with cells that received an equivalent amount of non-specific DNA (C; 100%). Experimental conditions include treatment with BmCAP-dsRNA and Luc-dsRNA at the indicated amounts (µg/ml in transfection mixture). N = 3. B. Functional assay after addition of nucleic acid (DNA or dsRNA) to the tissue culture medium. Cells were soaked with extracellular dsRNA for two days before transfection and for three days after transfection. Experimental conditions include treatment with DNA, BmCAP-dsRNA, BmHR3-dsRNA, Luc-dsRNA and GFP-dsRNA at the indicated concentrations (in µg/ml). Left: Luciferase assays to evaluate expression of the constitutive A-Luc transgene. Indicated is the luminescence/fluorescence ratio (N = 3). Right. Relative fluorescence measurements (fluorescence/luminescence ratios) 24 hrs after induction of the ecdysone ERE-gfp reporter with 200 nM of ecdysone agonist. Cells were soaked with nucleic acid (DNA or dsRNA) for two days before transfection and for another three days after transfection. At two days after transfection, cells were induced with hormone agonist for a period of 24 hours (N = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102679&req=5

pone-0020250-g003: RNAi functional assays.A. Functional assay after transfection of dsRNA. Bm5 cells were transfected with DNA and either non-specific dsRNA or specific dsRNA at different ratios in the absence (Left) or presence (Right) of TcR2D2 expression plasmid. Indicated is the amount of luminescence in comparison with cells that received an equivalent amount of non-specific DNA (C; 100%). Experimental conditions include treatment with BmCAP-dsRNA and Luc-dsRNA at the indicated amounts (µg/ml in transfection mixture). N = 3. B. Functional assay after addition of nucleic acid (DNA or dsRNA) to the tissue culture medium. Cells were soaked with extracellular dsRNA for two days before transfection and for three days after transfection. Experimental conditions include treatment with DNA, BmCAP-dsRNA, BmHR3-dsRNA, Luc-dsRNA and GFP-dsRNA at the indicated concentrations (in µg/ml). Left: Luciferase assays to evaluate expression of the constitutive A-Luc transgene. Indicated is the luminescence/fluorescence ratio (N = 3). Right. Relative fluorescence measurements (fluorescence/luminescence ratios) 24 hrs after induction of the ecdysone ERE-gfp reporter with 200 nM of ecdysone agonist. Cells were soaked with nucleic acid (DNA or dsRNA) for two days before transfection and for another three days after transfection. At two days after transfection, cells were induced with hormone agonist for a period of 24 hours (N = 3).
Mentions: It was observed that intracellular RNAi occurs efficiently in Bm5 cells in the absence of BmR2D2 expression. Co-transfection of the actin-luciferase reporter with Luc-dsRNA results in a dose-dependent reduction of luciferase activity (Figure 3A, left panel). This response is specific, since a high dose of a non-specific dsRNA (BmCAP-dsRNA) did not result in a decrease in luciferase activity. On the other hand, soaking of Bm5 cells with dsRNA in the extracellular medium at high concentration did not result in gene silencing (Figure 3B, right panel) as was observed before [22].

Bottom Line: It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues.Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells.The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

View Article: PubMed Central - PubMed

Affiliation: Insect Molecular Genetics and Biotechnology, Institute of Biology, National Centre for Scientific Research Demokritos, Athens, Greece. swevers@bio.demokritos.gr

ABSTRACT
RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

Show MeSH
Related in: MedlinePlus