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. 2004 Feb 19;23(4):876–884. doi: 10.1038/sj.emboj.7600096

Figure 4.

Figure 4

p19 suppresses RNA silencing in the heterologous in vitro RNA-silencing system. (A) p19 inhibits RNA silencing in the Drosophila in vitro system in a dose-dependent manner. In the RNA-silencing reaction, dsGFP350 dsRNA was used as inducer at 3 nM concentration and radioactively labelled full-length (725 nt) GFP transcript (200 pM) was added as a target RNA to the Drosophila embryo lysate. In vitro reactions were supplemented with various concentrations of CymRSV p19 expressed as a GST fusion protein (p19) or GST (87.5–2800 nM). Target degradation was monitored on a 1.2% agarose gel. RNA-silencing activity was indicated by loss of target RNA and appearance of the shorter degradation products (degraded target). (B) Effect of p19 on DICER activity in vitro. Radioactively labelled dsGFP350 was added to Drosophila embryo lysate at 3 nM concentration and p19 was used at various concentrations (21.87–2800 nM). dsRNA processing to siRNA was analysed on an 8% polyacrylamide gel. As a size marker, the Decade Marker System (Ambion) was used. (C) p19 inhibits RNA silencing by sequestering siRNAs in vitro. siRNA duplex (siGFP161) at 20 nM was used to induce the degradation of labelled ssGFP350 target RNA (100 pM) in the in vitro RNA-silencing system. Reactions were supplemented with various amounts of p19 (5.46–1400 nM final concentration). Inhibition of RNA-silencing activity is indicated by loss of the 74 base in the size 5′ end cleavage product. As a size marker, the Decade Marker System (Ambion) was used. (D) p19 cannot counteract with active RISC complex. Degradation of the ssGFP350 target RNA (lane 1) was induced by 20 nM siRNA (siGFP161, lane 2), or 500 mM ss siRNA (GFP161AS, lane 3). As indicated by the ±sign, 700 nM of p19 or GST was added simultaneously with siGFP161 or siGFP161AS to inhibit RNA silencing (lanes 4, 6, 7, 9). In lanes 5 and 8, p19 or GST was added 20 min after the addition of siGFP161. Inhibition of RNA silencing activity is indicated by loss of the 74 base in the size 5′ end cleavage product.