Our research is aimed at understanding the molecular mechanisms of RNA editing in kinetoplastid parasites, which cause African sleeping sickness, Chagas'disease, and leishmaniasis. Drug treatments for these diseases are antiquated, expensive, difficult to administer, often highly toxic, and resistance is developing. Thus, new chemotherapeutic agents to combat diseases caused by kinetoplastid parasites are urgently needed. Development of new drugs requires a detailed molecular understanding of parasite biology. Mitochondrial uridine insertion/deletion RNA editing is unique to kinetoplastids and absent from their human hosts. This process is essential for survival of both the human infective and insect vector forms of Trypanosoma brucei. Editing entails extensive remodeling of mRNAs by posttranscriptional insertion and deletion of uridine residues to create translatable mRNAs. Sequence information for editing is contained in small, trans acting guide RNAs (gRNAs), which act sequentially such that editing proceeds in a 3'to 5'direction along an mRNA. The RNA editing process is catalyzed by 20S editosomes. In addition, multiple accessory factors are required for productive editing of either subsets of RNAs or the entire pool of edited RNAs. During the previous funding period, we reported studies on TbRGG2, an essential protein that plays a key role in both the initiation and 3'to 5'progression of editing. TbRGG2 is a component of an ill-defined macromolecular complex, the mitochondrial RNA binding complex 1 (MRB1). Cells depleted of MRB1 components analyzed to date have remarkably different phenotypes with regard to gRNA and mRNA stability, the specific RNAs whose editing is affected, and the apparent point at which editing is compromised. Our overarching hypothesis is that the MRB1 complex is composed of subcomplexes whose combinatorial actions coordinate multiple fundamental steps in kinetoplastid RNA editing. In the proposed studies, we will employ a combined genetic, genomic, and biochemical approach to analyze the functions of the MRB1 complex and its constituent proteins in RNA editing in T. brucei.
In Aim 1, we will determine the roles of TbRGG2 RNA binding, annealing, and unwinding activities in editing initiation and 3'to 5'progression, and use deep sequencing and RNA structure analysis to define the specific intra- and intermolecular interactions that are overcome by TbRGG2.
In Aim 2, we will examine 11 previously unstudied MRB1 components using RNAi, followed by a battery of assays that will define the obstruction to complete editing in these cells. Finally, in Aim 3, we will determine the composition of the MRB1 complex in terms of its component subcomplexes and their protein- protein and protein-RNA interactions. Physical data from Aim 3 will be evaluated in the context of functional studies performed in Aims 1 and 2 to establish a comprehensive model of MRB1 complex structure and function. Together, these experiments will provide important insights into an essential gene regulatory process that may prove useful in the treatment of the deadly diseases caused by kinetoplastid parasites.
The long-term goal of this project is to understand the molecular mechanisms of uridine insertion/deletion RNA editing in kinetoplastid parasites. The proposed studies will significantly increase our mechanistic understanding of this essential gene regulatory process in a medically and economically important parasite. These studies may identify novel drug targets in kinetoplastids as well as provide insights into ribonucleoprotein function in higher eukaryotes.
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