Guide RNA Binding Complex

Boston University Medical Campus, Boston MA

Investigators

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Abstract

SUMMARY Parasitic infections caused by Trypanosoma brucei spp. have historically undermined public health and economies in Sub-Saharan Africa, only partially curtailed by recently approved therapeutics. Although the long- term impact of repurposed drugs remains unclear, sustained research has positioned T. brucei among the most prolific unicellular model systems. Paradigms of antigenic variation, GPI-anchoring, RNA interference, and others have critically informed eukaryotic biology, while discoveries of RNA editing and guide RNAs (gRNAs) have translated into genome and transcriptome-altering technologies. The trypanosomal mitochondrial editosome consists of interacting RNA binding and enzymatic modalities. RNA editing substrate binding complexes (RESCs) enclose gRNAs and mRNAs, initiating editing by scaffolding their imperfect hybrid. RNA editing catalytic complexes (RECCs) recognize mismatches in the hybrid as editing sites and execute mRNA cleavage, uridine insertion or deletion, and re-ligation reactions. A principal auxiliary factor, DEAH-box RNA helicase KREH2, likely contributes to editing progression, although the mechanism remains unclear. Empowered by our recent advances in cryogenic electron microscopy (cryoEM) of mitochondrial ribonucleoproteins and macromolecular enzymes, we propose visualizing the editing pathway by determining RESC structures at discernible steps. Through charting protein composition, interactions, and RNA occupancy, we will define the functions of distinct RESCs and determine their structures by cryoEM. Genetic and reconstitution approaches will validate structural predictions and align RESC remodeling and editing events. Aim 1 investigates RESCs that bind gRNA and pre-mRNA to initiate and monitor editing and evict gRNA upon completion. We have discovered and characterized commensurate RESC states. We will solve their structures, identify RNA cargo and interactions, and reconstitute proteinaceous components in a heterologous host. Aim 2 examines KREH2 helicase as the putative ATP-dependent molecular motor driving the editing pathway by remodeling RESCs. We hypothesize that KREH2 recognizes the gRNA’s 3′ end and disassembles RESCs by translocating the gRNA 5′-bound to RESC2 pseudo-triphosphatase. By modulating KREH2 and RESC2 activities, we will interrogate RESC permutations and concomitant RNA dynamics.