Site-specific and mRNA-specific control of accurate mRNA editing by a helicase complex in trypanosomes

Abstract

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 co-purify with editing mRNAs but the functional interplay between these complexes remains unclear. Most steady-state mRNAs are partially edited and include mis-edited ‘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 non-canonical 2U-insertion that causes major pausing in 3’-5’ progression. In A6, a non-canonical 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.