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 system 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 including the RNA-free RNA editing core complex (RECC) and auxiliary ribonucleoprotein (RNP) complexes. 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 ( H2 F1). Both of these 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, H2 F1 recruits REH2 to the editing apparatus, and H2 F1 downregulation causes REH2 fragmentation. Our systematic mutagenesis dissected determinants in REH2 and H2 F1 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 that impact the assembly and interactions of REH2C. H2 F1 upregulation stabilized REH2 in vivo . Mutation of the core cysteines or basic amino acids in individual zinc fingers affected the stabilizing property of H2 F1 but not its interactions with other examined editing components. This result suggests that 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.

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

“…Differences in the cell lines used (e.g., inoculum quality or growth rate) for each sample group, or our manipulation of the samples, may account for the variations in cumulative values between the two sample groups. Possible differences in REH2C protein subunit stoichiometry, as previously suggested after sedimentation analysis of mitochondrial extracts (Kumar et al 2019), may also cause cumulative differences between KREH2-IP and mtRNA samples versus KH2F1-IP and mtRNA samples. Notably, total editing in most ESs in the 5' half (approximately ES45-to-ES72) was relatively stable compared to the gradual decline in total editing in the 3' half.…”

“…As above, the different cell lines for each group of samples or our manipulations of the samples may account for the variations in cumulative values between the two sample groups. Possible variations in REH2C protein subunit stoichiometry, as suggested above (Kumar et al 2019), could also cause cumulative differences between KREH2-IP and mtRNA samples versus KH2F1-IP and mtRNA samples. Several prominent spikes in Inc/Cor appeared to contribute to the differences between the cumulative curves of these samples.…”

“…The function of the KREH2 subunit in the REH2C complex is of particular interest. This ATP-dependent RNA helicase of the DEAH/RHA family exhibits a conserved multi-domain organization in related helicases from yeast to humans (Cruz-Reyes et al 2016;Kumar et al 2019). KREH2 is the sole DEAH box helicase in trypanosomal editosomes However, spliceosomes, and ribosome biogenesis require multiple DExH/RHA helicases with a variety of functions (Jarmoskaite and Russell 2014).…”

“…Independent biological replicates of each sample (n= 2) were prepared from separate cultures, including RNAiinduced and an untreated control samples. Mitochondria-enriched extracts were prepared as reported (Kumar et al 2019). Total RNA from mitochondrial extract (mtRNA) was isolated using TRIzol per manufacturer's instructions.…”

“…RESC is heterogeneous and its organization is not completely understood, but it contains gRNA and at least 14 protein subunits (Hashimi et al 2008;Panigrahi et al 2008;Weng et al 2008;Ammerman et al 2012). REH2C includes three protein subunits and ATP-dependent 3'-5' double-stranded RNA unwinding activity (Madina et al 2015;Kumar et al 2016;Kumar et al 2019). The kinetoplastid RNA helicase KREH2 (aka REH2) and zinc-finger protein KH2F1 (aka H2 F1) subunits of REH2C promote efficient editing (Hashimi et al 2009;Kumar et al 2016).…”