Analysis of edited mRNA coverage by gRNAs in Illumina libraries. (A–G) Steady-state gRNA from PF parasites and gRNA in immunopurified MRBs are annotated in blocks of edited sequence, each directed by a gRNA. Cumulative standard and G·U pairs between edited mRNA and gRNAs are scored in nucleotide frequency plots (NFPs Log2), including initiating gRNAs (i.e., block 1 or B1) and major upstream gRNAs (B2 or B3) in our libraries. Some gRNAs may guide alternative editing (e.g., B1.alt and B2.alt), and the ND7 5′ domain uses at least two similar initiating gRNAs (B1a and B1b). The entire editing domain in cytochrome B (CYb) and the 3′ terminus of other domains are plotted. The sequence of CYb (including all 34 U-insertions as lowercase t's) and the ND7 5′ domain are shown. Equal protein loads were applied to the IPs, and the gRNA was gel-isolated and extracted. gRNA from IPs and total RNA were adjusted to apply comparable amounts in the libraries (e.g., Supplemental Fig. S3; see Materials and Methods). gRNA from the IPs was directly ligated to the adapters, whereas total gRNA from PF parasites was first treated with 5′ monophosphate specific Terminator exonuclease, which degrades rRNA but not 5′ triphosphate gRNA ends. (H,I) Northern blots of select initiating gRNAs from IPs and PF cells. The blot was hybridized with the A6 gRNA probe, stripped, and re-used with the ND7 gRNA probe. 15% UREA-PAGE was run as in Figure 1C.
