| In kinetoplastid RNA editing, uridylate residues are inserted and deleted from numerous, specific sites within mitochondrial transcripts by a complex with seven major, and several critical but minor staining components. Two of the major proteins, bands-IV and -V, were shown to be RNA ligases. However, roles for the other major proteins were unknown. Using cells depleted of band-II, band-VI, or band-VII by RNA interference, I assessed the function of these editing components.; My characterization of band-II focused on its role in editing catalysis. By individually assessing the editing steps, Catherine Huang and I demonstrated this protein is required for each step of U-insertion, but no steps of U-deletion. Analysis of basic enzymatic activities revealed that band-II is none of the U-insertional enzymes and instead functions by providing substrate and/or protein recognition for those enzymes.; Using extracts from band-VII RNAi cells, I assessed the role of this protein in editing complex association, protein retention, and editing catalysis. In collaboration, this protein was shown critical for editing complex integrity. I further determined that without the complex, the other editing proteins are destabilized and slowly diminish in abundance. My editing analyses demonstrate that band-VII is critical for both U-deletional and U-insertional cleavages, possibly because they also depend on a nearly complete editing complex. Similarly, I assessed the role of band-VI. This protein, although not required for editing complex integrity, augments its stability. The other major proteins remain and actively catalyze the final two steps of U-deletion and U-insertion. However, band-VI is required for U-deletional cleavage, and is a critical editing site-specificity factor for the U-insertional endonuclease.; My use of both specific and basic editing assays to discern loss of an activity from loss of editing substrate recognition, along with optimization of such assays for use with rapidly prepared extracts, which retain activities lost from traditionally prepared extracts, was key in determining the roles of these essential proteins. Results presented in this thesis contribute significantly to the current view of the editing complex as functionally and spatially partitioned and further demonstrate that the editing enzymes require auxiliary factors to function at editing sites. |
