FUNCTIONAL CHARACTERIZATION OF MRB8180-CONTAINING COMPLEXES IN TRYPANOSOME RNA EDITING
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Trypanosoma brucei is the causative agent of Human African Trypanosomiasis and nagana in cattle. Additionally, two close cousins to this parasite, T. cruzi and species of Leishmania, also cause disease in humans. Collectively, these organisms are found on every continent except for Antarctica and considered Neglected Tropical Diseases. Studying the basic biology of these parasites provides valuable information for drug design efforts as parasite-specific biology has the potential be an effective drug target with minimal effects on the host. Uridine insertion/deletion RNA editing is an essential, parasite-specific process that is found in all kinetoplastids. Through this process, mitochondrial mRNAs are modified by specific insertion and deletion of uridines. Editing generates functional open reading frames that encode mitochondrial respiratory proteins. Both enzymatic and non-enzymatic factors are required for RNA editing and much is still unknown about how this process proceeds and how it is regulated. The roles of numerous non-enzymatic factors have remained opaque in part due to the limitations of conventional methods to interrogate the order and mechanism by which editing progresses and thus the roles individual proteins play in mediating this progression. To overcome this limitation, I developed a novel bioinformatic platform, the Trypanosome RNA Editing Alignment Tool (TREAT), which allows us to examine whole populations of partially edited sequences using high throughput sequencing. In this thesis, I examine whole populations of partially edited mRNA in wild type procyclic cells. We determined that there are Intrinsic Pause Sites (IPSs) in the editing process and that these likely arise by the mechanism distinct from that which gives rise to the Major Junction End Sites (MJESs). The MJESs are enriched after deletion actions, suggesting that the switch from deletion to insertion action is more limited than going from insertion to deletion. We also foundevidence of potentially translatable alternative editing and extensive variation in the 5’UTRs of mitochondrial mRNA. In the second part of this work, I used HTS/TREAT to elucidate the roles of three proteins in the RNA Editing Mediator Complex (REMC). We determined that the three factors examined function in the progression of editing through a gRNA, rather than in gRNA exchange. However, they have distinct roles, and REMC is likely heterogeneous in composition. MRB8170/4160 plays a transcript-specific role in editing initiation, and constrains the region of active editing. In contrast, TbRGG2 and MRB8180 are required for editing to proceed through a gRNA-defined region in a manner that does not go strictly site-by-site (i.e., non-linear), presumably due to their ability to promote gRNA/mRNA plasticity. In this thesis, I provide the first evidence that editing can proceed through distinct pathways, i.e.the order in which a region is modified, and that non-linear modifications are essential to the editing process, generating com-monly observed junction regions. Additionally, I provide evidence that the HTS/TREAT methodology can greatly illuminate recalcitrant parts of the RNA editing process. My data support a model in which RNA editing is executed via multiple pathways that necessitate successive re-modification of junction regions facilitated, in part, by the REMC variant containing TbRGG2 and MRB8180.