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.
