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. ? ? Our research aims to unravel the functioning of the editosome at the atomic level by crystallographic methods to ultimately: ? (i) obtain a full understanding of its architecture; ? (ii) unravel the substrate specificity of each editosomal enzyme; ? (iii) elucidate key interactions of the guide RNA:pre-mRNA duplex with the editosomal proteins; ? (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. ? ? 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. ? ? 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. ? ? ?
