High-throughput sequence analysis of mitochondrial transcripts. (A) Average number of normalized pre-edited ND7-5′, RPS12, and CYb transcripts for eight uninduced (Avg Unin) and two MRB10130 RNAi induced samples (MRB10130 KD). P-values (unpaired t-test with Welch's correction): 0.0183 for ND7-5′ (*), <0.0001 for RPS12 (****), and 0.0149 for CYb (*). (B) RPS12 3′ edited mRNA sequence with exacerbated pause sites (EPSs) arising upon MRB10130 RNAi in both biological replicates indicated with red diamonds. Black diamonds represent previously reported EPSs arising upon MRB7260 knockdown (McAdams et al. 2018). No EPSs were detected 5′ of the region shown in either cell line. Gray bars below the sequence indicate gRNAs, numbered 3′ to 5′ (Koslowsky et al. 2014). gRNA anchor regions are depicted with bold lines. Hatched regions at the 5′ and 3′ ends represent variation in gRNA lengths within a gRNA class. RPS12 stop codon is underlined; additional underscores are shown for clarity in stretches of unedited sequence to align numbers with the correct ES. Lower case u indicates a U inserted by editing; asterisks indicate encoded U's deleted by editing. (C) EPSs arising in CYb mRNA upon knockdown of MRB10130 and MRB7260. Shown is the edited domain and flanking sequence. The start codon is underlined, and the underscore indicates ES 580. All symbols as in B. (D) Sequences of the most abundant junctions within the region directed by RPS12 gRNAs 1–3 in MRB10130 replete and depleted cells. These sequences span editing stop sites (ESS) 10–40. Average normalized counts of a given sequence in uninduced samples (AvgUn) and MRB10130 knockdown (KD) are ordered from highest to lowest fold change upon MRB10130 knockdown. ESS refers to the editing stop site for that sequence. Jcn indicates the number of ES spanned by a given junction sequence. Fold indicates the fold change in the number of sequences for each junction in MRB10130 RNAi samples compared to uninduced samples. Pre-edited sequence, black; canonically edited sequence, red; junctions, purple. Gray lines below pre-edited and edited sequences indicate gRNA-directed blocks, with anchor regions depicted as bold lines. ES are indicated with triangles and are numbered from 3′ to 5′. Red diamonds; EPSs that arise following MRB10130 depletion.
