RNA editing in trypanosome mitochondria: Biochemical mechanism and identifying components of the ribonucleoprotein editing complex

he mitochondrial mRNAs of trypanosomes are often post-transcriptionally modified by an RNA processing event, termed RNA editing, which results in the insertion or deletion of uridylate (U) residues in mRNAs. RNA editing is necessary for the formation of complete coding sequences for several essential mitochondrial proteins. The number and site of U addition and deletion is directed by small guide RNAs (gRNAs). The biochemical mechanism as well as the proteins required to catalyze this unusual RNA processing event was unknown and this was the focus of my project. I distinguished between two proposed models of kinetoplastid RNA editing: transesterification versus enzymatic cleavage-ligation. I was able to identify two mitochondrial RNA ligases (50 and 57 kDa) which catalyze RNA ligation via a three step reaction pathway, including an adenylylated enzyme intermediate. This adenylylated form of RNA ligase was shown to be involved in the formation of proposed chimeric RNA editing intermediates. The involvement of RNA ligase in the in vitro production of editing intermediates provided strong evidence for a cleavage-ligation mechanism of kinetoplastid RNA editing.;Next I illustrated RNA ligase involvement in in vitro U-deletional and U-insertional RNA editing reactions. By following this essential editing complex component, RNA ligase, I then purified editing RNPs from both T. brucei and C. fasciculata. T. brucei mitochondrial extracts were fractionated by cation-exchange, anion-exchange, affinity and size-exclusion chromatography. The final purified fraction exists as a ribonucleoprotein particle (RNP) of...