1. Site-specific and mRNA-specific control of accurate mRNA editing by a helicase complex in trypanosomes
- Author
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Jorge Cruz-Reyes, Andrew Hillhouse, Daniel Camilo Osorio Hurtado, Alasdair Ivens, Xiuren Zhang, Pawan K. Doharey, Joshua Meehan, Zachary T Goodall, Achim Schnaufer, Vikas Kumar, and James J. Cai
- Subjects
Trypanosoma ,kinetoplastid RNA editing ,RNA, Mitochondrial ,Protein subunit ,Trypanosoma brucei brucei ,Protozoan Proteins ,Article ,Substrate Specificity ,03 medical and health sciences ,Ribosomal protein ,Animals ,Nucleotide ,Guide RNA ,RNA, Messenger ,RNA-Seq ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Messenger RNA ,biology ,030302 biochemistry & molecular biology ,Helicase ,RNA ,Cell biology ,editosome guide RNA ,chemistry ,RNA processing ,RNA editing ,biology.protein ,RNA Editing ,RNA, Protozoan ,trypanosoma brucei ,RNA, Guide, Kinetoplastida ,REH2C helicase complex - 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 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.
- Published
- 2020