Our long-term goal is to understand the molecular mechanisms that control RNA editing during the life cycle of Trypanosoma brucei. Kinetoplastid protozoa include important pathogenic species of Trypanosoma, Leishmania and Phytomonas. These ancient flagellates undergo a dramatic form of mitochondrial mRNA maturation by insertion and deletion of U residues, which can create over half of the protein-coding sequence. In T. brucei, editing is differentially regulated in the insect vector and the mammalian host (procyclic """"""""Pf"""""""" and bloodstream """"""""Bf"""""""" forms, respectively). Studying unique biological processes, such like this, may suggest ways of halting the development cycle and hence the progress of the disease. While the mechanism of Pf editing is known and the protein composition of Pf editing complexes is starting to emerge, fundamental questions remain unexplored including the function and composition of Bf complexes, and the relevant pre-mRNA/protein interactions during Pf and Bf editing.
Our specific aims are (i) to apply our newly established RNA editing system in bloodstream (Bf) trypanosomes to study regulation in vitro, and (ii) to identify pre-mRNA/ protein interactions during Pf and Bf editing. My lab recently established a long-sought in vitro system to study Bf full editing by U insertion and U deletion. Our preliminary data show significant differences between Pf and Bf complexes and their editing activity. We also began characterizing pre-mRNA/ protein crosslinks that co-purify with editing complexes upon high-stringency affinity chromatography.
In Aim 1 we propose to further compare the protein composition and function of Bf and Pf complexes, search for differential protein components, and identify editing steps and pre-mRNA features involved in regulation.
In Aim 2, we propose to further characterize two current crosslinking proteins, particularly their physical and/or functional relationship with editing complexes. We will similarly characterize additional RNA/protein interactions. Our studies compare stage-specific and constitutive premRNAs (paired or not to gRNA) under catalytic conditions. We expect to characterize RNA/protein contacts relevant for function, which may be stage-specific and/or transcript-specific. Together these studies should provide important insights into the developmental control of RNA editing, and understanding of relevant RNA/protein interactions during this unique maturation process. ? ?
