Date Published: April 29, 2019
Publisher: Public Library of Science
Author(s): Vikas Kumar, Pawan K. Doharey, Shelly Gulati, Joshua Meehan, Mary G. Martinez, Karrisa Hughes, Blaine H. M. Mooers, Jorge Cruz-Reyes, Monika Oberer.
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 (H2F1). 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, 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 that impact the assembly and interactions of REH2C. H2F1 upregulation stabilized REH2 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. 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.
Kinetoplastid protozoa including Trypanosoma brucei are early-branching eukaryotes that require extensive insertion and deletion of uridylates in most mitochondrial mRNAs (reviewed in ). This RNA editing is protein-catalyzed and directed by small guide RNAs (gRNAs) with complementarity to fully-edited mRNA. Most editing progresses in blocks that typically overlap and each block is directed by a single gRNA . The editing machinery includes multiple subcomplexes: the ~20S catalytic RNA editing core complex (RECC, also called the 20S editosome) and auxiliary editing RNPs. The ~15S REH2-associated complex (REH2C) characterized here includes RNA editing helicase 2 (REH2), REH2-associated protein factor 1 (H2F1), H2F2, and mRNA substrates and products [3, 4]. However, only REH2 and H2F1 participate in editing. REH2C interacts with a larger accessory editing RNP complex termed the RNA editing substrate binding complex (RESC), which also contains mRNA [5, 6]. The presence of mRNA in RESC was confirmed by others [7, 8].
Previous studies revealed that the native REH2C exhibits RNA-mediated interactions with at least two variants of RESC [3–5]. These variants contain gRNA and comparable levels of GAP1 and RGG2, that typify GRBC and REMC, respectively. However, these variants differ minimally in their relative content of the canonical MRB3010 in GRBC. Additional variations in protein composition have been reported in purifications of RESC components by different labs (reviewed in ). Interestingly, native and genetically-induced changes in MRB3010 stoichiometry may be common. While the precise reasons for these changes are unclear, we reasoned that they reflect relevant dynamic changes in the assembly and function of the editing apparatus. In this study, we examined protein features that affect the formation of REH2C, its stable association with RESC components, and its transient contacts with the RECC enzyme. Because the examined helicase-associated RESC variant exhibits substoichiometric MRB3010, we refer to its variant GRBC module as GRBC* . Using antibodies made available to us, we have detected other proteins in pulldowns of REH2 that may be part of this RESC variant including both GAP1/GAP2 paralogs and MRB8170. The REH2 pulldowns also contain MRB6070 that was found in a MRB6070/MRB1590 subcomplex that associates via RNA with RESC (S1 Fig). However, further analysis of relative composition between variants is needed.
In the current study, we examined over 20 constructs of REH2 and H2F1 (Tables 1 and 2) to identify features in these editing proteins that impact the stability of REH2, the assembly of the REH2C RNP, and its RNA-mediated coupling with other components of the RNA editing holoenzyme (or RNA holo-editosome). We also formally established that the isolated REH2 is a bona fide RNA helicase enzyme.