Figure 2.

Characterisation of p19-bound 21-nt dsRNAs. (A) Analyses of p19-bound 21-nt RNAs by quantitative Northern blotting using increasing amounts of a synthetic 21-nt oligonucleotide complementer to the positive strand of CymRSV and RNA prepared from α-p19 IP (Figure 1D). An internally labelled positive strand of CymRSV was used as a probe. (B) Quantification of data obtained in (A). Closed circles, concentration standards; open circle, RNA isolated from α-p19 IP. The line shows the linear fit of the standards calculated with the computer program Microcal Origin 5.00. (C) 21-nt dsRNA from α-p19 IP driving the degradation of the cognate target RNA in the in vitro RNA-silencing system. A final concentration of 16.38–262.2 nMp19-bound 21-nt dsRNAs was added to the reactions. For target RNA, we used the full-length CymRSV₁₋₄₇₃₃ transcript at 100 pM concentration. dsRNA (5 nM) corresponding to the full-length CymRSV was used as a positive control. For negative control, the same system except GFP target RNA at 200 pM and dsGFP350 at 3 nM in lane 2 was used.