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AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system.

Schuck J, Gursinsky T, Pantaleo V, Burgyán J, Behrens SE - Nucleic Acids Res. (2013)

Bottom Line: This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC.These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC.The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biotechnology (NFI), Martin Luther University Halle-Wittenberg, Halle/Saale D-06120, Germany.

ABSTRACT
AGO/RISC-mediated antiviral RNA silencing, an important component of the plant's immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (-)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.

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Antiviral RNA silencing with a virus-derived siRNA pool. (A) ‘RISC formation/cleavage assay’ with DI-R3.5 RNA. A pool of siRNAs was generated by RNase III (ShortCut®) cleavage of dsR3.5 RNA. Using BYL where AGO1 was overexpressed by in vitro translation, RISC was formed with this siRNA pool. 32P-labeled (+) or (−)DI-R3.5 RNA transcripts were added to the extract that contained the programmed RISC, and the reaction products were subsequently analyzed by denaturing PAGE and autoradiography (lanes 2 and 4). The analogous experiments performed with a non-related (‘gf698’) siRNA served as negative controls (lanes 1 and 3). The positions of the labeled target RNAs are indicated; most prominent cleavage products are indicated by asterisks. (B) Schematic representation of the in vitro ‘replication inhibition assay’. A BYL reaction mixture that contained in vitro translated AGO1 and RISC that was ‘programmed’ with the siRNA(s) of choice was added to a second BYL reaction mixture that contained the in vitro translated TBSV replicase proteins p33 and p92. In experimental ‘variant 1’, RNA replication was initiated by combining both reactions and by the subsequent addition of replication mix and DI-R3.5 RNA template. In experimental ‘variant 2’, the mixture that contained the programmed RISC was added at a later time point to the replication reaction (Figure 4E). (C) ‘replication inhibition assay’. The reaction described in (B) was performed with (+)DI-R3.5 RNA. Lane 1; in the absence of p92 (no replication). Lane 2; with an unrelated (‘gf698’) siRNA (negative control). Lane 3; with the dsR3.5-generated vsiRNA pool. The RNA replication products (RP) are indicated, as well as the most prominent RNA cleavage products (asterisks).
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gkt193-F3: Antiviral RNA silencing with a virus-derived siRNA pool. (A) ‘RISC formation/cleavage assay’ with DI-R3.5 RNA. A pool of siRNAs was generated by RNase III (ShortCut®) cleavage of dsR3.5 RNA. Using BYL where AGO1 was overexpressed by in vitro translation, RISC was formed with this siRNA pool. 32P-labeled (+) or (−)DI-R3.5 RNA transcripts were added to the extract that contained the programmed RISC, and the reaction products were subsequently analyzed by denaturing PAGE and autoradiography (lanes 2 and 4). The analogous experiments performed with a non-related (‘gf698’) siRNA served as negative controls (lanes 1 and 3). The positions of the labeled target RNAs are indicated; most prominent cleavage products are indicated by asterisks. (B) Schematic representation of the in vitro ‘replication inhibition assay’. A BYL reaction mixture that contained in vitro translated AGO1 and RISC that was ‘programmed’ with the siRNA(s) of choice was added to a second BYL reaction mixture that contained the in vitro translated TBSV replicase proteins p33 and p92. In experimental ‘variant 1’, RNA replication was initiated by combining both reactions and by the subsequent addition of replication mix and DI-R3.5 RNA template. In experimental ‘variant 2’, the mixture that contained the programmed RISC was added at a later time point to the replication reaction (Figure 4E). (C) ‘replication inhibition assay’. The reaction described in (B) was performed with (+)DI-R3.5 RNA. Lane 1; in the absence of p92 (no replication). Lane 2; with an unrelated (‘gf698’) siRNA (negative control). Lane 3; with the dsR3.5-generated vsiRNA pool. The RNA replication products (RP) are indicated, as well as the most prominent RNA cleavage products (asterisks).

Mentions: The in vitro replication assay was performed essentially as described previously (46). That is, the TBSV p33 and p92 proteins were generated by in vitro translation in the BYL using the described conditions and 5 pmol of p33 mRNA and 0.25 pmol of p92 mRNA in a 50-µl reaction. For replication, 40 µl of the translation reaction was mixed with 10 µl of 5× RdRp buffer (50 mM DTT, 500 µg/ml of actinomycin D, 17 mM magnesium acetate, 5 mM of each ATP, GTP and UTP and 0.250 mM of CTP containing 15 µCi of [α-32P]CTP). In all, 0.5 pmol of TBSV DI RNA or of the respective DI RNA variants was added as a template, and the reaction was performed for 3 h at 25°C. Total RNA was purified, and the 32P-labeled RNA products were analyzed as described previously. To test for RISC-mediated antiviral RNA silencing, the reaction volume of the in vitro translation reaction that generated p33 and p92 was reduced to 25 µl. In a parallel 25-µl reaction, AGO mRNA (2 µg) was translated in the presence of the siRNA that was used to ‘program’ the assembling RISC. If not indicated differently (see text and Figure 3B), both translation reactions were combined and replication initiated via the addition of RdRp buffer and DI RNA. The replication products were analyzed as described previously. Suppression of antiviral RNA silencing was achieved via the addition of 25 U of purified p19 (New England Biolabs) to the translation reaction that generated AGO1 and RISC.


AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system.

Schuck J, Gursinsky T, Pantaleo V, Burgyán J, Behrens SE - Nucleic Acids Res. (2013)

Antiviral RNA silencing with a virus-derived siRNA pool. (A) ‘RISC formation/cleavage assay’ with DI-R3.5 RNA. A pool of siRNAs was generated by RNase III (ShortCut®) cleavage of dsR3.5 RNA. Using BYL where AGO1 was overexpressed by in vitro translation, RISC was formed with this siRNA pool. 32P-labeled (+) or (−)DI-R3.5 RNA transcripts were added to the extract that contained the programmed RISC, and the reaction products were subsequently analyzed by denaturing PAGE and autoradiography (lanes 2 and 4). The analogous experiments performed with a non-related (‘gf698’) siRNA served as negative controls (lanes 1 and 3). The positions of the labeled target RNAs are indicated; most prominent cleavage products are indicated by asterisks. (B) Schematic representation of the in vitro ‘replication inhibition assay’. A BYL reaction mixture that contained in vitro translated AGO1 and RISC that was ‘programmed’ with the siRNA(s) of choice was added to a second BYL reaction mixture that contained the in vitro translated TBSV replicase proteins p33 and p92. In experimental ‘variant 1’, RNA replication was initiated by combining both reactions and by the subsequent addition of replication mix and DI-R3.5 RNA template. In experimental ‘variant 2’, the mixture that contained the programmed RISC was added at a later time point to the replication reaction (Figure 4E). (C) ‘replication inhibition assay’. The reaction described in (B) was performed with (+)DI-R3.5 RNA. Lane 1; in the absence of p92 (no replication). Lane 2; with an unrelated (‘gf698’) siRNA (negative control). Lane 3; with the dsR3.5-generated vsiRNA pool. The RNA replication products (RP) are indicated, as well as the most prominent RNA cleavage products (asterisks).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3643602&req=5

gkt193-F3: Antiviral RNA silencing with a virus-derived siRNA pool. (A) ‘RISC formation/cleavage assay’ with DI-R3.5 RNA. A pool of siRNAs was generated by RNase III (ShortCut®) cleavage of dsR3.5 RNA. Using BYL where AGO1 was overexpressed by in vitro translation, RISC was formed with this siRNA pool. 32P-labeled (+) or (−)DI-R3.5 RNA transcripts were added to the extract that contained the programmed RISC, and the reaction products were subsequently analyzed by denaturing PAGE and autoradiography (lanes 2 and 4). The analogous experiments performed with a non-related (‘gf698’) siRNA served as negative controls (lanes 1 and 3). The positions of the labeled target RNAs are indicated; most prominent cleavage products are indicated by asterisks. (B) Schematic representation of the in vitro ‘replication inhibition assay’. A BYL reaction mixture that contained in vitro translated AGO1 and RISC that was ‘programmed’ with the siRNA(s) of choice was added to a second BYL reaction mixture that contained the in vitro translated TBSV replicase proteins p33 and p92. In experimental ‘variant 1’, RNA replication was initiated by combining both reactions and by the subsequent addition of replication mix and DI-R3.5 RNA template. In experimental ‘variant 2’, the mixture that contained the programmed RISC was added at a later time point to the replication reaction (Figure 4E). (C) ‘replication inhibition assay’. The reaction described in (B) was performed with (+)DI-R3.5 RNA. Lane 1; in the absence of p92 (no replication). Lane 2; with an unrelated (‘gf698’) siRNA (negative control). Lane 3; with the dsR3.5-generated vsiRNA pool. The RNA replication products (RP) are indicated, as well as the most prominent RNA cleavage products (asterisks).
Mentions: The in vitro replication assay was performed essentially as described previously (46). That is, the TBSV p33 and p92 proteins were generated by in vitro translation in the BYL using the described conditions and 5 pmol of p33 mRNA and 0.25 pmol of p92 mRNA in a 50-µl reaction. For replication, 40 µl of the translation reaction was mixed with 10 µl of 5× RdRp buffer (50 mM DTT, 500 µg/ml of actinomycin D, 17 mM magnesium acetate, 5 mM of each ATP, GTP and UTP and 0.250 mM of CTP containing 15 µCi of [α-32P]CTP). In all, 0.5 pmol of TBSV DI RNA or of the respective DI RNA variants was added as a template, and the reaction was performed for 3 h at 25°C. Total RNA was purified, and the 32P-labeled RNA products were analyzed as described previously. To test for RISC-mediated antiviral RNA silencing, the reaction volume of the in vitro translation reaction that generated p33 and p92 was reduced to 25 µl. In a parallel 25-µl reaction, AGO mRNA (2 µg) was translated in the presence of the siRNA that was used to ‘program’ the assembling RISC. If not indicated differently (see text and Figure 3B), both translation reactions were combined and replication initiated via the addition of RdRp buffer and DI RNA. The replication products were analyzed as described previously. Suppression of antiviral RNA silencing was achieved via the addition of 25 U of purified p19 (New England Biolabs) to the translation reaction that generated AGO1 and RISC.

Bottom Line: This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC.These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC.The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biotechnology (NFI), Martin Luther University Halle-Wittenberg, Halle/Saale D-06120, Germany.

ABSTRACT
AGO/RISC-mediated antiviral RNA silencing, an important component of the plant's immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (-)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.

Show MeSH
Related in: MedlinePlus