Regulation of RNA editing in two life cycle stages of Trypanosoma brucei

Pathogenic kinetoplastid protozoa, including Trypanosoma brucei, exhibit a remarkable mechanism of gene expression control by RNA editing. In most organisms, RNA is copied from DNA and directs protein synthesis without changes in the code. However, in trypanosome mitochondria (the ‘powerhouse’ of cells), mRNAs are remodeled by massive addition or removal of uridine residues. In their complex life cycle, trypanosomes alternate between two very different hosts: mammals (including humans) and insects, which are the vectors of transmission. The dramatically different environments that the parasites face in humans and insects demand rapid and large-scale metabolic and physiological changes, including in RNA editing. This process is catalyzed by the editosome and directed by anti- sense guide RNAs (gRNAs). The crucial question of how the RNA editing mechanism is precisely regulated with some mRNAs undergoing full editing maturation in one stage but not the other during parasite development remains unanswered and is the focus of this research. Prior studies have shown that REH2C controls editing fidelity in editosomes by unclear mechanisms. Here, our multidisciplinary team will join efforts to test a novel REH2C-based model of T. brucei editing regulation during development. Completion of the proposed studies will provide insights into a question that has remained a mystery for decades: What are the key proteins and mechanisms in differential RNA editing across the T. brucei life cycle? Our model will be tested in bloodstream form (BSF) and insect procyclic form (PCF) parasites. Transcriptomics and genomics will be combined to examine major early control checkpoints, including use of novel repressive “moonlighting” gRNAs (Aim 1), and genome-wide global control of gRNA utilization (Aim 2). A structural modeling and homology-guided approach will be combined with a powerful genetic toolbox that we established for this proposal to examine candidate REH2C protein determinants in editing regulation (Aim 3). All Aims include novel concepts and complementary tools, including REH2C gain- and loss-of-function BSF and PCF cell lines. While all three Aims are complementary, they can be executed independently to examine a long-standing question in trypanosomal RNA biology.