Working model. Dynamic higher-order MRB1 “organizers” composed of several specialized subcomplexes (MRBs) with different RNA/protein composition may coordinate recruitment of editing substrates (pre-edited mRNA and gRNA) and catalytic RECCs forming an editing holoenzyme. MRBs may bind edited mRNAs and route them into ribosomes by undefined “hand-over” mechanisms. Native helicase REH2 binds RNA, presumably gRNA, mRNA, or both, and forms a stable MRB with editing substrates, unwinding activity, and several subunits including other RNA-binding proteins (stars). REH2-associated unwinding seems to promote stable assembly of editing substrates with REH2-MRB but could also affect other MRBs and editing progression. The MRB3010 subunit affects an early editing step and may not bind RNA directly but forms a separate stable MRB. The current study showed that the MRB3010-subcomplex associates with editing substrates and edited mRNA and suggests that it promotes early editing through its preferential recruitment of initiating gRNAs. Both purified REH2-MRB and MRB3010-MRB contain GAP1/2 subunits that bind and stabilize gRNA. A common RNA-binding core in MRBs may include GAP proteins and several RNA crosslinking subunits that we detected but need to be identified. Dynamic interactions between these MRBs and additional factors (MRBs and non-MRBs not shown in the cartoon) may control substrate recruitment and interactions during initiation and progression between blocks (e.g., B1-to-B4). A proposed TbRGG2 MRB-like subcomplex does not contain REH2, MRB3010, or GAP proteins (Madina et al. 2011; Ammerman et al. 2012). Distinct RNA/protein composition of MRBs may also impact transient associations with RECCs, mitoribosomes, and other mitochondrial factors. Recombinant proteins known to crosslink with synthetic RNA (stars) include the following: GAPs with gRNA, and the 4160/8160 paralogs and TbRGG2 preferentially with mRNA-like fragments. The initiation gRNA anchor anneals just 3′ of the editing domain. Later anchors often need edited sequence. Whether gRNAs remain annealed to edited mRNA is unknown.
