The C-terminus of the KREH1 helicase is important for RNA binding and association with mitochondrial RNA editing complexes in T. brucei

Abstract

Mitochondrial pre-mRNA editing in kinetoplastids, a clade that includes human-infective parasitic protists such as Trypanosoma and Leishmania, is required to produce translatable, functional gene products from otherwise nonfunctional mitochondrial precursor transcripts of essential respiratory chain complex subunits. RNA editing is a template-guided process in which cognate guide RNAs (gRNAs) anneal to specific sites at the pre-mRNA and direct the complementary insertion and/or deletion of uridines. Large multi-modular complexes - like the RNA-Editing Substrate-binding Complex (RESC) and the RNA-Editing Catalytic Complexes (RECC) - organize and catalyze RNA editing, respectively. Within these assemblies, Kinetoplast RNA Editing Helicases (KREHs) play an important role in ribonucleoprotein complex remodeling, particularly in pan-editing, when transcripts are extensively modified throughout their length by multiple gRNAs. However, the mechanisms by which KREH RNA helicases facilitate pan-editing, and their specific roles in this process, remain unclear. Here, we define the molecular properties of the Trypanosoma brucei DEAD-box RNA editing helicase KREH1. Cellular co-proteome analyses reveal that KREH1 interacts with subunits of the RESC complex and biochemical experiments show that KREH1 is an RNA-dependent ATPase that unwinds double-stranded RNA substrates. We show that the flexible C-terminus of KREH1 is important for RNA binding and, consequently, for enzymatic activity in vitro and interactions with the RESC complex in vivo. Overall, our experiments refine the role of KREH1 in the pan-editing process and establish its utility as a molecular tool for investigating higher-order RNA editing complexes in the cellular context.