Fig. 2. A→I editing progressively inhibits RNAi and production of siRNAs. (A) CAT dsRNA was edited to varying degrees (5, 12, 19, 25 and 43% A→I) using ADAR2 and analysed by native gel electrophoresis. As the level of editing increased there was a corresponding reduction in their mobility on a native gel (for example, compare lanes 1 and 6). Each deaminated RNA was relatively homogeneous, as indicated by the relatively tight bands with lower mobility than unmodified dsRNA. (B) An RNAi assay was performed for 90 min where each CAT d-dsRNA was tested for its ability to activate RNAi (lanes 2–6) alongside dsRNA (lane 1) and ssRNA (lane 7). In this assay the control RNA used was ΔKP. As the level of editing increased in the d-dsRNAs, there was a clear reduction in the amount of RNAi (compare lanes 1 and 6). (C) The data from (B) are represented as the CAT/ΔKP ratio relative to the CAT/ΔKP ratio in the presence of ssRNA. In order to accurately quantitate the amount of target RNA remaining in the presence of highly edited d-dsRNA (lanes 4–6), it was necessary to determine the sum of the two upper bands that correspond to CAT RNA (marked with an asterisk in lane 6). The anomalous mobility of a small proportion of the CAT target RNA is the result of partial hybridization of the CAT target RNA with the 200-fold excess of unlabelled CAT d-dsRNA. An inverse correlation exists between the level of editing in d-dsRNA and its ability to induce RNAi. (D) The production of siRNAs from CAT dsRNA and d-dsRNAs was analysed using RNAs labelled with [α³²P]ATP. This assay was performed alongside that shown in (B) using identical conditions. As the level of editing increased, the production of siRNAs showed a corresponding decrease (compare lanes 1 and 6). The percentage of the input RNA that was converted to siRNAs is shown below each lane.