Twenty-four species of kinetoplastids (Trypanosoma and Leishmania) infect 30 million people worldwide and threaten 600 million people with debilitating and sometimes fatal infections. Kinetoplastids have a unique RNA editing system (kRNA editing) not found in humans, rendering a promising drug target. No one has effectively why non-canonical base pairing is a conserved feature of the binary mRNA-gRNA complex that is central to RNA editing in trypanosomes. Addressing this issue with structure-function- based studies is important because it is the first step in the rational design of drugs against the RNA editing complex. Our long-term goals are to understand the role of RNA structure in kRNA editing and more generally to relate sequence with structure. The immediate goal of this project is to determine how non-canonical RNA base pairs alter the structure and function of the U-helix domain of the pre- mRNA/gRNA complexes. Our central hypothesis is that non-canonical base pairs affect RNA editing by influencing the width of the major groove and by affecting the thermal stability of the double helix. The widening of the major groove may enhance recognition by proteins in the editosome and reduced thermal stability may allow absorption of torsional stain introduced by strand separation at the editing site before endonucleolytic cleavage of the pre-mRNA strand, and it may facilitate the progression of editing from the first editing site to th second editing site along the pre-mRNA strand. We test the hypothesis using structure-function studies of single and multiple point mutations in the U-helix.
In aim 1, we test the effect of non-canonical base pairs near the editing sites in the template domain and in the U-helix domain on editing efficiency in vitro.
In aim 2, we determine the effects of these non-canonical base pairs on the thermodynamic stability of the gRNA/mRNA duplex.
In aim 3, we will determine the effects of non-canonical base pairs on the structure of the gRNA/mRNA duplex by X-ray crystallography. More accurate models of dsRNA-an expected outcome of this project-will contribute to design of inhibitors that target kRNA editing in trypanosomes but also impact other important areas such as 1) general design of dsRNAs including therapeutic RNAs, 2) more accurate predictions of RNA structure, and 3) simulations of RNA folding. The outcomes of this project are expected to advance in our understanding of the role of non-canonical base pairing in RNA editing. This advancement in understanding will aid the development of more effective therapies to fight infections with trypanosomes.
This project will determine the effect of RNA three-dimensional structure on the RNA editing complexes that are unique to the mitochondria of trypanosomes. The new knowledge gained from this project will provide critical data for the effort to improve the health of millions of people infected with these pathogenic parasites by developing safer and more effective treatments of trypanosome infections such as Chagas disease and sleeping sickness.
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