Role of RNA structure in developmental RNA editing regulation in trypanosomes

Pathogenic kinetoplastids, including Trypanosoma brucei, exhibit a remarkable mechanism of gene expression control by RNA editing. For most organisms, RNA is copied from DNA and directs protein synthesis without changes in the code. In trypanosome mitochondria (mt), the ‘powerhouse’ of cells, mRNAs require massive addition or removal of uridine residues in two lifecycle stages: bloodstream forms (BSF) in mammalian hosts and procyclic forms (PCF) in insect vectors. The different environments faced by T. brucei in humans and insects demand rapid and large-scale metabolic and physiological changes, including RNA editing. Most mt-mRNAs required editing at hundreds of sites directed by many anti-sense guide RNAs (gRNAs) in multi-RNP editosomes. The crucial question of how RNA editing is developmentally regulated, including the role of RNA structure, remains unanswered. This significant, long-standing knowledge gap is the focus of this proposal. A multidisciplinary team will join efforts to test a model of editing regulation involving RNA conformation and regulatory proteins. Editing is energetically demanding, so early regulation is expected. Our preliminary studies identified major early checkpoints in three mRNAs where gRNA-directed alternative (non-canonical) editing creates a high-frequency element (HFE). These studies plus initial dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) of synthetic mRNAs revealed that HFE formation at early checkpoints (a) blocks canonical editing, (b) installs conformation that may “attenuate” editosomes, and (c) is modulated by REH2C proteins in a stage-specific fashion. Our in vitro DMS chemical probing suggests that these checkpoints involve repressive RNA determinants. However, intracellular conditions, including developmental stages or RNP association, may impact RNA structure. To examine this model, Aim 1 will develop targeted mitoDMS-MaPseq to test the hypothesis that HFE-mediated structure is impacted in vivo vs. in vitro. We have already established optimal conditions for DMS reactivity in trypanosome mitochondria and examined the RNA structure of synthetic mRNAs folded in vitro. We will first analyze HFE transcripts folded in native conditions (PCF vs. BSF total mt-RNA) versus in vitro. This first approach may reveal if in-cell or developmental stage affects RNA topology at checkpoints. Aim 2 will test the hypothesis that RNA conformation at early checkpoints is impacted by ribonucleoprotein complexes (RNPs). We established that two holo-editosome RNPs, RESC and REH2C, bind mRNA and that HFE formation can be enhanced in RESC and modulated by REH2C. In this Aim, we will determine (a) DMS reactivity at early checkpoints in these immunopurified RNPs vs. total mt-RNA (from Aim 1), (b) whether depletion or overexpression of REH2C proteins impact DMS reactivity at early checkpoints in total mt-RNA and RESC. Completion of these studies will determine whether native conditions, including developmental stage, RNP binding, or the level of regulatory proteins in REH2C, impact RNA conformation at early checkpoints. Our studies include innovative concepts and technological advances in trypanosome mitochondria to address a central open question in developmental RNA editing regulation.