Structure-Function Relationships in Kinetoplastid RNA-Editing

University Of Washington, Seattle WA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): RNA editing in kinetoplastids, several of which are global pathogens, is a unique, and essential, process in the mitochondria of these ancient eukaryotes. This process uses hundreds of so-called "guide RNAs" to edit an incomplete "pre-messenger RNA" by U-insertion and deletion at hundreds of specific editing sites. Many more U's are inserted than deleted. In some cases, "pan-editing" occurs which can more than double the size of the pre-message. A central role in this U-insertion/deletion editing is played by the "editosome". This is an assembly of approximately 20 nuclear-encoded proteins including six different RNA editing enzymes performing a precisely orchestrated sequence of RNA cleavage, insertion/deletion and religation reactions. [unreadable] [unreadable] Our research aims to unravel the functioning of the editosome at the atomic level by crystallographic methods to ultimately: [unreadable] (i) obtain a full understanding of its architecture; [unreadable] (ii) unravel the substrate specificity of each editosomal enzyme; [unreadable] (iii) elucidate key interactions of the guide RNA:pre-mRNA duplex with the editosomal proteins; [unreadable] (iv) discover the large conformational changes the protein and RNA molecules must undergo while the pre-message is growing by the action of the six different enzymes. [unreadable] [unreadable] Our proposal builds on recent successes including the crystal structure determinations of the RNA Editing Ligase 1 catalytic domain and that of the editing 3'-Terminal-Uridylylate Transferase. In the latter case the lone pair of an exquisitely positioned water molecule appears to be the key to U-specificity. A subcomplex of three different editosomal proteins has been obtained which appears to be a heterohexamer. These initial results provide an excellent platform from which to proceed with unraveling the many remaining mechanistic mysteries of this marvelous U-insertion/deletion machinery. [unreadable] [unreadable] Since several editing proteins are essential in pathogenic kinetoplastids, the structures we plan to determine are also promising starting points for the design of selective inhibitors of key pathogen proteins. [unreadable] [unreadable] [unreadable]