Mechanistic Studies Of Rna Editing In Trypanosomes

Author : Vaibhav Mehta
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (119 download)

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Download or read book Dissecting RNA Editing Mechanism "in Vitro" with Genetic and Chemical Genetic Approaches written by Vaibhav Mehta and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: "Trypanosoma brucei, Trypanosoma cruzi and the Leishmania species are the major pathogenic trypanosomatid organisms that cause devastating diseases in humans and animals, with high incidence and mortality rates. These organisms create functional mitochondrial transcripts by RNA editing, a process catalyzed by multiprotein editosome complexes that entail insertion and/or deletion of uridylates (Us) as dictated by short complementary guide RNAs and associated accessory proteins. RNA editing is essential for the parasite’s viability, making the process an attractive target for drug development. However, how the editosome proteins assemble and function together is not well understood. The focus of this thesis is on identifying small molecule inhibitors of RNA editing as chemical tools to dissect editosome assembly and as lead compounds against the trypanosomatid pathogens. First, using our FRET-based RNA editing assay, we screened the library of 1280 pharmacologically active compounds against RNA editing and identified new inhibitors of this process. Four compounds showed significant potency inhibiting RNA editing, with IC50 values ranging from 1 to 5 [mu]M. We found that to a varying extent, they interfered with RNA-protein interaction. Additionally, we showed for the first time that a compound identified in this screen, NF449, an analog of suramin, could effectively kill T. brucei in vitro. While NF449 does not specifically block a desired catalytic core enzyme and seems to interfere with RNA-protein interaction, these data establish feasibility and proof of concept for finding potent inhibitors of trypanosome RNA editing.In the next part of my thesis, we investigate the naphthalene-based inhibitors that competitively inhibit the auto-adenylylation activity of the recombinant kinetoplastid RNA editing ligase 1 (KREL1). In the context of purified editosomes, the inhibitors block all catalytic editing activities by interfering with RNA-protein interactions, similar to the pilot-scale screen hits. Through mass spectrometry analyses, we identify the accessory mitochondrial RNA binding proteins, MRP1 and MRP2, as potential off-targets for these compounds, and demonstrate their inhibitory activity on recombinant MRP1/2 heterotetramer complex. The compounds affect the association between the core editosomes and MRP1/2 heterotetramer. Functional editosomes purified post-treatment with the compounds exhibit compromised in vitro RNA editing activity that, remarkably, recovers upon addition of recombinant MRP1/2 proteins, directly implicating an essential role for MRP1/2 in the initiation of RNA editing. Next, to evaluate the molecular details of KREL1-inhibitor interactions and binding selectivity, we performed alanine replacement mutagenesis of recombinant KREL1 protein on residues predicted to bind the inhibitors. We identified the residues critical for binding to facilitate the design of more potent inhibitors. Furthermore, as KREL1 activity is enhanced by its interacting protein partner, A2, characterizing the interaction between these two proteins benefits structure-activity exploration of inhibitors. In the last part of my thesis, we examined this interaction using full-length, truncated, and point-mutated KREL1. Our analyses narrow down the point of A2 contact to the terminal 59 residues of KREL1 and identify critical residues not only in this region but also in the ATP binding domain that play a mechanistic role in enhancing ligase activity.The studies described here contribute significantly to functional organization and assembly of the editosome proteins and identify the regulatory mechanisms for the essential mitochondrial gene expression in these organisms. Furthermore, the introduced compounds can be starting points for the development of critically needed anti-trypanosomatid drugs against these disease agents"--