One of the most fascinating forms of RNA processing reactions occurs within the kinetoplastid protozoa. Several mRNAs of the mitochondria within this order must be edited through the precise insertion and deletion of uridine nucleotides. Part of the genetic information specifying the location and nature of editing is contained within guide RNAs (gRNAs). A specific gRNA is able to bind to its cognate mRNA immediately 3' of a block of editing sites and also contains a guide sequence complementary to a block of edited mRNA sequence. Changes to the guide sequence result in corresponding changes to the number of uridine nucleotides inserted and deleted from the mRNA. The long-term objective of this project has been to understand the role of both gRNA and mRNA sequence and structure on the editing reaction. An A+U rich sequence within the 5' untranslated region of a cytochrome b mRNA impedes the fidelity of in vitro gRNA-directed editing, is sufficient to induce editing when inserted within a normally unedited transcript, and also appears to affect in vivo editing. A highly similar sequence is found within the cytochrome b gRNA, and mutation of this sequence activates in vitro gRNA-directed editing. Taken together, the results suggest that the A?U rich sequence either directly interacts with the editing machinery or is closely associated with it.
The first aim of the proposal is to identify the interacting A+U binding protein, and the second aim is to determine how it is released from the RNAs, enabling the machinery to act at editing sites; several biochemical and genetic approaches will be used. Accomplishing these aims will identify a mechanism through which kinetoplastid editing is regulated. Included within the kinetoplastid order are several species of Leishmania and Trypanosoma that are some of the most devastating human parasites. Since inhibition of editing has been demonstrated to be lethal to the parasites, defining a regulatory mechanism for the reaction will identify several targets for the development of new drugs.
