Wednesday, February 5, 2025
2/5/2025
SUMMARY Parasitic infections by Trypanosoma brucei spp. undermine public health and economy in Sub-Saharan Africa. From the basic science vantage point, discoveries of antigenic variation, trans-splicing, and RNA editing in these early branching protozoans have contributed fundamental tenets of eukaryotic biology. Notably, work on mitochondrial U-insertion/deletion mRNA editing introduced the guide RNA concept, a foundation of modern genome altering technologies. Identification of terminal RNA uridylyltransferases (TUTases) in trypanosomes has led to recognition of uridylation’s broad significance and diverse cellular roles, ranging from microRNA turnover in flies to maternal mRNA clearance in mammals. Emerging structural insights establish mitochondrial U-tailing in T. brucei as the paradigm of RNA processing and decay pathways present in most eukaryotes. Conversely, the internal U-insertion is a central editing reaction unique to Kinetoplastea. This project will reveal the mechanisms of TUTase-dependent RNA maturation and editing processes at atomic resolution. Aim 1 investigates how the TUTase—exonuclease—RNA helicase nexus orchestrates mitochondrial RNA metabolism. It focuses on protein complexes that couple U-tailing by KRET1 (T1) with degradation, and thereby govern biogenesis, functionality, and metabolic fates of rRNAs, mRNAs and guide RNAs. Molecular, proteomics, imaging and cryo-EM approaches will define T1 complexes’ functions, composition, interactomes and structures. We have identified and will characterize an RNA helicase which links gRNA processing and editing. Aim 2 examines the structural basis of U-insertion mRNA editing. We have discovered KRET2 (T2) and MEAT1 (M1) and their distinct complexes. By determining in vivo substrates and cryo-EM structures of these assemblies, we will uncover the mechanisms of editing site selection and U-insertion by divergent T2 and M1 TUTases.
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