Uridine insertion/deletion RNA editing is an essential process in kinetoplastid parasites whereby mitochondrial mRNAs are modified through the specific insertion and deletion of uridines to generate functional open reading frames, many of which encode components of the mitochondrial respiratory chain. The roles of numerous non-enzymatic editing factors have remained opaque given the limitations of conventional methods to interrogate the order and mechanism by which editing progresses and thus roles of individual proteins. Here, we examined whole populations of partially edited sequences using high throughput sequencing and a novel bioinformatic platform, the Trypanosome RNA Editing Alignment Tool (TREAT), to elucidate the roles of three proteins in the RNA Editing Mediator Complex (REMC). We determined that the factors examined function in the progression of editing through a gRNA; however, they have distinct roles and REMC is likely heterogeneous in composition. We provide the first evidence that editing can proceed through numerous paths within a single gRNA and that non-linear modifications are essential, generating commonly observed junction regions. Our data support a model in which RNA editing is executed via multiple paths that necessitate successive re-modification of junction regions facilitated, in part, by the REMC variant containing TbRGG2 and MRB8180.

Trypanosoma brucei undergoes an essential process of mitochondrial uridine insertion and deletion RNA editing catalyzed by a 20S editosome. The multiprotein mitochondrial RNA-binding complex 1 (MRB1) is emerging as an equally essential component of the trypanosome RNA editing machinery, with additional functions in gRNA and mRNA stabilization. The distinct and overlapping protein compositions of reported MRB1 complexes and diverse MRB1 functions suggest that the complex is composed of subcomplexes with RNA-dependent and independent interactions. To determine the architecture of the MRB1 complex, we performed a comprehensive yeast two-hybrid analysis of 31 reported MRB1 proteins. We also used in vivo analyses of tagged MRB1 components to confirm direct and RNA-mediated interactions. Here, we show that MRB1 contains a core complex comprised of six proteins and maintained by numerous direct interactions. The MRB1 core associates with multiple subcomplexes and proteins through RNA-enhanced or RNA-dependent interactions. These findings provide a framework for interpretation of previous functional studies and suggest that MRB1 is a dynamic complex that coordinates various aspects of mitochondrial gene regulation.

In the mitochondria of kinetoplastid protozoa, including Trypanosoma brucei, RNA editing inserts and/or deletes uridines from pre-mRNAs to produce mature, translatable mRNAs. RNA editing is carried out by several related multiprotein complexes known as editosomes, which contain all of the enzymatic components required for catalysis of editing. In addition, noneditosome accessory factors necessary for editing of specific RNAs have also been described. Here, we report the in vitro and in vivo characterization of the mitochondrial TbRGG2 protein (originally termed TbRGGm) and demonstrate that it acts as an editing accessory factor. TbRGG2 is an RNA-binding protein with a preference for poly(U). TbRGG2 protein levels are up-regulated 10-fold in procyclic form T. brucei compared with bloodstream forms. Nevertheless, the protein is essential for growth in both life cycle stages. TbRGG2 associates with RNase-sensitive and RNase-insensitive mitochondrial complexes, and a small fraction of the protein co-immunoprecipitates with editosomes. RNA interference-mediated depletion of TbRGG2 in both procyclic and bloodstream form T. brucei leads to a dramatic decrease in pan-edited RNAs and in some cases a corresponding increase in the pre-edited RNA. TbRGG2 downregulation also results in moderate stabilization of never-edited and minimally edited RNAs. Thus, our data are consistent with a model in which TbRGG2 is multifunctional, strongly facilitating the editing of pan-edited RNAs and modestly destabilizing minimally edited and never-edited RNAs. This is the first example of an RNA editing accessory factor that functions in the mammalian infective T. brucei life cycle stage.Trypanosoma brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in African wildlife. During their life cycle, T. brucei are transmitted between two different hosts, the tse tse fly insect vector and mammalian host. Due to the resulting drastic changes in environmental growth conditions, the parasite displays differentiationdependent mechanisms of energy metabolism. In the insect midgut stage (procyclic form (PF) 3 ), energy is generated through cytochrome-mediated oxidative phosphorylation, whereas in the mammalian bloodstream form (BF), energy is generated strictly through glycolysis. Correspondingly, the single mitochondrion of T. brucei undergoes extensive alterations in both morphology and gene expression during differentiation. One aspect of this mitochondrial gene regulation is uridine insertion/deletion RNA editing, a process that is restricted to kinetoplastid protozoa, of which T. brucei is a member. During this process, uridine residues are posttranscriptionally added to and/or deleted from pre-mRNAs to produce translatable mature mRNAs. In T. brucei, the accumulation of many edited RNAs is life cycle stage-dependent, with RNAs encoding the cytochrome components apocytochrome b (CYb) and cytochrome oxidase subunit II (COII) edited only in PF and editing of components of the NADH dehydrogenase complex up-regulated i...