TbRGG2 differentially affects editing of pan-edited RPS12 RNA at the 3′ and 5′ ends. (A) Diagram of fully edited RPS12 RNA depicting the nucleotide position (top) and the number of edited sites (ESs; bottom) on the fully edited RNA. The regions of the RNA where editing is examined using qRT-PCR and poisoned primer extensions (PPEs) are labeled. (B) Representative initiation PPE. The reaction was performed using radiolabeled primers that anneal to the 3′ end of RPS12 RNA from PF TbRGG2 RNAi cells after 72 h with (I) or without (U) tetracycline induction. The products arising from extension of the edited (E) RNAs are labeled. Tubulin RNA levels (TUB) were analyzed by PPE in the same reaction as a control. (C) Representative 3′ end PPE. The reaction was performed using radiolabeled primers that anneal to the 3′ end of RPS12 RNA from PF TbRGG2 RNAi cells after 72 h with (I) or without (U) tetracycline induction. The products arising from extension of unedited (UE) and edited (E) RNAs are labeled. Tubulin RNA levels (TUB) were analyzed by PPE in the same reaction as a control. (D) Comparison of the decrease in edited RNA levels following TbRGG2 loss using qRT-PCR to analyze 5′ fully edited transcripts (Fisk et al. 2008) and PPEs to analyze editing at initiation and near the 3′ ends of the transcripts. For the PPE reactions, the decreases in edited RNA levels were determined using RNA isolated from at least duplicate experiments. Edited RNA levels were normalized to tubulin PPEs performed in the same reaction.
