MRB7260 is essential for productive protein–RNA interactions within the RNA editing substrate binding complex during trypanosome RNA editing

Abstract

The trypanosome NAditing ubstrate bindingomplex (RESC) acts as the platform for mitochondrial uridine insertion/deletion RNA editing and facilitates the protein-protein and protein-RNA interactions required for the editing process RESC is broadly comprised of two subcomplexes: GRBC (uide NAinding omplex) and REMC (NA ditingediator omplex). Here, we characterize the function and position in RESC organization of a previously unstudied RESC protein, MRB7260. We show that MRB7260 forms numerous RESC-related complexes, including a novel, small complex with the guide RNA binding protein, GAP1, which is a canonical GRBC component, and REMC components MRB8170 and TbRGG2. RNA immunoprecipitations in MRB7260-depleted cells show that MRB7260 is critical for normal RNA trafficking between REMC and GRBC. Analysis of protein-protein interactions also reveals an important role for MRB7260 in promoting stable association of the two subcomplexes. High-throughput sequencing analysis of RPS12 mRNAs from MRB7260 replete and depleted cells demonstrates that MRB7260 is critical for gRNA exchange and early gRNA utilization, with the exception of the initiating gRNA. Together, these data demonstrate that MRB7260 is essential for productive protein-RNA interactions with RESC during RNA editing.

MRB10130 is a RESC assembly factor that promotes kinetoplastid RNA editing initiation and progression

“…This includes COII mRNA, whose editing does not depend on trans-acting gRNAs or GAP1/ 2. Because the effects of editing factor depletion are typically more dramatic on pan-edited than on minimally edited mRNAs (Fisk et al 2008;Acestor et al 2009;Ammerman et al 2011Ammerman et al , 2013Huang et al 2015;Simpson et al 2017;McAdams et al 2018), these data suggest a distinct and fundamental function for MRB10130. To determine whether the MRB10130 impact on editing could involve direct RNA binding, we performed UV cross-linking assays with recombinant MRB10130 and body-labeled gRNA or mRNA.…”

“…We can then further analyze editing progression by determining the editing stop site in each mRNA, which is defined as the final (5 ′ most) ES that matches the canonical fully edited sequence correctly (Table 1; Simpson et al 2016). At a population level, TREAT allows us to define exacerbated pause sites (EPSs; Table 1); EPSs are editing stop sites at which canonical editing pauses to a significant extent in a given knockdown cell line compared to uninduced controls (P < 0.05, q < 0.05) (Simpson et al 2017;McAdams et al 2018;Tylec et al 2019). Additionally, we can analyze the lengths and sequences of misedited junctions 5 ′ of EPSs to provide insights into specific editing defects that arise when distinct editing factors are depleted (Table 1; Simpson et al 2017;McAdams et al 2018;Tylec et al 2019).…”

“…Early proteomic and yeast two-hybrid studies identified two modules comprising RESC, the guide RNA binding complex (GRBC) and the RNA editing mediator complex (REMC), that appear to associate in a dynamic fashion mediated in part by protein-RNA interactions (Ammerman et al 2012;Aphasizheva et al 2014). More recent studies point to the existence of numerous variants of RESC (Kafkova et al 2012;Madina et al 2015;Simpson et al 2017;McAdams et al 2018), and the organization and functions of RESC are a subject of ongoing research.…”

“…For example, the founding and defining component of this complex is the GAP1/2 heterotetramer, which is essential for stability of the entire gRNA population (Weng et al 2008;Hashimi et al 2009;Kumar et al 2016). GAP1/2 is canonically referred to as a component of the GRBC module; however, it has now been identified in small complexes apart from fully assembled RESC that contain either REH2, TbRGG3, or MRB7260, indicating a broader function for this protein than first appreciated (Madina et al 2014;McAdams et al 2015McAdams et al , 2018. Several components of the REMC module have been studied using biochemical assays, domain interaction and protein-RNA interaction studies, and high-throughput sequencing (HTS), in one case combined with in vivo protein-RNA crosslinking.…”

“…We recently showed that the RESC component, MRB7260, plays a role in modulating GRBC-REMC interactions within the RESC complex during editing (McAdams et al 2018). Depletion of MRB7260 caused dissociation of GRBC and REMC, with concomitant disruption of normal mRNA and gRNA trafficking within RESC.…”