KREH2 helicase represses ND7 mRNA editing in procyclic-stageTrypanosoma bruceiby opposite modulation of canonical and “moonlighting” gRNA utilization creating a proposed mRNA structure

Abstract

Unknown factors regulate mitochondrial U-insertion/deletion (U-indel) RNA editing in procyclic-form (PCF) and bloodstream-form (BSF) trypanosomes. This editing is directed by anti-sense gRNAs, creates canonical protein-encoding mRNAs, and may developmentally control respiration. Canonical editing by housekeeping cognate gRNAs occurs amid massive non-canonical editing of unclear sources and biological significance. We found PCF-specific repression in mRNA ND7 that involves novel modulation of gRNA usage by RNA helicase KREH2. At a major early checkpoint near the mRNA 3' terminus, KREH2 promotes the usage of a non-cognate "terminator" gRNA over a cognate gRNA. At this early checkpoint, terminator-directed editing installs a structural element in 30% of the ND7 transcriptome, which potentially occludes the mRNA 3' terminus and represses further editing. BSF-to-PCF differentiation in vitro recreated this form of negative control. Remarkably, KREH2-RNAi knockdown relieved repression by reverting the native usage of cognate/non-cognate gRNAs at the major early checkpoint. ND7 transcripts that lacked terminator-directed editing at the early checkpoint exhibited similar negative editing control along the mRNA sequence, suggesting that KREH2 modulates guiding fidelity globally. Thus, KREH2 is a key protein in ND7 developmental editing control involving modulation of gRNA selection and RNA structure installed by a regulatory terminator gRNA.