Development of precleaved deletion and insertion assays. Assays are monitored using radiolabeled HHR RNA substrates (top panel) and FRET-based HHR substrates (bottom panel) with several controls (as described in the middle panel). (A) PC-deletion assay and (B) PC-insertion assay with all components except functional editosome (lane 1), guide RNA (lane 2) and 3′ fragment (lane 3). Both assays in the presence of RNA editing inhibitors (MrB and suramin, lanes 4 and 5). A faint product/signal was detected without ATP (lane 6). In the presence of all reaction components with discernible product/signal (lane 7). Improved HHR activity is observed when ATP addition is delayed by 1-h post reaction initiation (lane 8). Active HHR (0.5 and 1.5 pmol) was used as a size and activity control in the PC-insertion and deletion assay, respectively, to magnify the enhancement in activity (lane 9). Means and standard deviations in the bottom plot were obtained from four replicates. Lanes 6,7, and 8 in both panels were significantly different from no protein controls (lane 1) (P < 0.05) by one-way ANOVA (GraphPad Prism).
