RNA Uridylation in Trypanosomes

摘要

ABSTRACT Parasitic infections by Trypanosoma brucei spp. undermine public health and economy in Sub-Saharan Africa. These hemoflagellates are distinguished by the kinetoplast, a mitochondrial nucleoprotein body containing interlinked maxicircle and minicircle DNA. From a basic knowledge perspective, findings of polycistronic transcription, trans-splicing and RNA editing in trypanosomatids have profoundly influenced the conceptual fabric of RNA biology. Accordingly, ongoing work on U-insertion/deletion mitochondrial mRNA editing has produced pivotal breakthroughs, including discoveries of gRNA, the editosome, and the first RNA uridylating enzyme, KRET1 TUTase. Sustained efforts by the PI and other laboratories have expanded the uridylation’s breadth from a unique editing reaction into a compound transcriptome-shaping force prominent in most eukaryotes. Building on recently unraveled mitochondrial and cytosolic mRNA maturation and decay pathways, and progress in TUTase structural biology, we submit these Specific Aims: Aim 1 investigates the TUTase–3′-5′ exonuclease nexus underpinning mitochondrial RNA biogenesis and degradation. It focuses on mechanisms by which KRET1-containing molecular machines recognize diverse ribonucleoprotein particles, thus governing processing, functions, and metabolic fates of their cargo RNA. Molecular and Cryo-EM approaches will define transcript type-specific KRET1 complexes and determine their interactomes, in vivo RNA targets and near-atomic structures. We will analyze a novel RNA helicase, KREH3, as the potential link between massive antisense transcription of kinetoplast DNA and KRET1-mediated turnover. Aim 2 examines structural determinants and interactions enabling KRET2 and MEAT1 to recognize double- stranded RNA substrates, such as gRNA–mRNA hybrids. These programable mitochondrial TUTases execute U-insertion editing, which constitutes a Kinetoplastea-specific method of genetic information transfer. Aim 3 explores TbTUT3 and TbTUT4 as potential modulators of cytosolic mRNA decay, a principal conduit regulating nuclear gene expression in lieu of missing transcriptional control. By assessing transcriptome-wide U- tailed mRNAs prevalence and decay rates, we will test the coupling between mRNA 3′ uridylation and directional clearance. Beyond the parasite RNA metabolism, we are inquiring how the divergent auxiliary modalities confer orthogonal specificities and functions to a conserved and phylogenetically pervasive TUTase catalytic domain.