Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes

Abstract

Uridylate insertion/deletion RNA editing in Trypanosoma brucei is a complex system that is not found in humans, so there is interest in targeting this system for drug development. This systeme uses hundreds of small non-coding guide RNAs (gRNAs) to modify the mitochondrial mRNA transcriptome. This process occurs in holo-editosomes that assemble several macromolecular trans factors around mRNA: the RNA-free RNA editing core complex (RECC) and auxiliary RNPs. Yet, the regulatory mechanisms of editing remain obscure. The enzymatic accessory RNP complex, termed the REH2C, includes mRNA substrates and products, the multi-domain 240 kDa RNA Editing Helicase 2 (REH2) and an intriguing 8-zinc finger protein termed REH2-Associated Factor 1 (H2F1). Both proteins are essential in editing. REH2 is a member of the DExH/RHA subfamily of RNA helicases with a conserved C-terminus that includes a regulatory OB-fold domain. In trypanosomes, H2F1 recruits REH2 to the editing apparatus, and H2F1 downregulation causes REH2 fragmentation. Our systematic mutagenesis dissected determinants in REH2 and H2F1 for the assembly of REH2C, the stability of REH2, and the RNA-mediated association of REH2C with other editing trans factors. We identified functional OB-fold amino acids in eukaryotic DExH/RHA helicases that are conserved in REH2 and impact the assembly and interactions of REH2C. H2F1 upregulation stabilized the large REH2 polypeptide in vivo. Mutation of the core cysteines or basic amino acids in individual zinc fingers affected the stabilizing property of H2F1 but not its interactions with other examined editing components. Thus, most if not all fingers may contribute to REH2 stabilization. Finally, a recombinant REH2 (240 kDa) established that the full-length protein is a bona fide RNA helicase with ATP-dependent unwinding activity. REH2 is the only DExH/RHA-type helicase in kinetoplastid holo-editosomes.