Trypanosome U-insertion/deletion RNA editing in mitochondrial mRNAs involves guide RNAs (gRNAs) and the auxiliary RNA editing substrate binding complex (RESC) and RNA editing helicase 2 complex (REH2C). RESC and REH2C stably copurify with editing mRNAs but the functional interplay between these complexes remains unclear. Most steady-state mRNAs are partially edited and include misedited “junction” regions that match neither pre-mRNA nor fully edited transcripts. Editing specificity is central to mitochondrial RNA maturation and function, but its basic control mechanisms remain unclear. Here we applied a novel nucleotide-resolution RNA-seq approach to examine ribosomal protein subunit 12 (RPS12) and ATPase subunit 6 (A6) mRNA transcripts. We directly compared transcripts associated with RESC and REH2C to those found in total mitochondrial RNA. RESC-associated transcripts exhibited site-preferential enrichments in total and accurate edits. REH2C loss-of-function induced similar substrate-specific and site-specific editing effects in total and RESC-associated RNA. It decreased total editing primarily at RPS12 5′ positions but increased total editing at examined A6 3′ positions. REH2C loss-of-function caused site-preferential loss of accurate editing in both transcripts. However, changes in total or accurate edits did not necessarily involve common sites. A few 5′ nucleotides of the initiating gRNA (gRNA-1) directed accurate editing in both transcripts. However, in RPS12, two conserved 3′-terminal adenines in gRNA-1 could direct a noncanonical 2U-insertion that causes major pausing in 3′–5′ progression. In A6, a noncanonical sequence element that depends on REH2C in a region normally targeted by the 3′ half of gRNA-1 may hinder early editing progression. Overall, we defined transcript-specific effects of REH2C loss.
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