Determinants of Specificity for the Trypanosoma brucei A to I tRNA Editing Deaminase

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The Ohio State University

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Trypanosomatids are responsible for causing illness and death among millions of humans and animals worldwide. Two main genera of these unicellular flagellated parasites are Trypanosoma, which cause African sleeping sickness and Chagas disease, and Leishmania, which cause Leishmaniasis. The World Health Organization estimates that approximately 400 million people across the globe are at risk of infections caused by these protozoa. In addition to their pathogenic characteristics, trypanosomatids also represent some of the earliest mitochondria-containing eukaryotes. Trypanosomatids exhibit many significant and interesting biological processes, such as an extreme degree of RNA editing (as with U insertion and deletion in mRNAs) and complete tRNA import into the mitochondria, which is important for mitochondrial protein synthesis. In translation, although there are 61 amino acid codons, there is only a small subset of tRNAs to decode them. As a result, some flexibility is needed to allow for a single tRNA to decode multiple codons—as proposed by Francis Crick with the Wobble hypothesis in the 1960s (1). One nucleotide capable of increasing pairing flexibility during decoding is the nucleotide inosine, which in the third position of the tRNA anticodon, allows recognition of up to three different nucleotides in an mRNA codon: uridine, cytosine, and adenosine. The editing event that leads to inosine formation in tRNAs, is catalyzed by a heterodimeric enzyme known as ADAT2/3 (adenosine deaminases acting on tRNA). Remarkably, inosine is not encoded in DNA and is only found in RNAs as a result of an enzyme-mediated reaction, which is essential for viability. The goal of this project was to analyze the effects various mutations at the anticodon stem loop of tRNAThr have on inosine formation at the first (wobble) position of the anticodon of tRNAs in Trypanosoma brucei. We hypothesized that mutations at these positions of the tRNA will decrease deamination efficiency and substrate recognition by ADAT2/3. Through in vitro methods, which include protein-RNA binding assays (band-shift assays) and enzyme kinetics the effect of mutations on ADAT2/3 function was evaluated. These results could shed light on how ADAT2/3 binds tRNA and catalyzes the conversion of adenosine to inosine. In addition, these observations can also lead to future design of therapeutics against parasites of such major medical importance (2). 1. Crick, FH. (1966). Codon—anticodon pairing: the wobble hypothesis. J Mol Biol. 19, 548-555. 2. Gerber, A. P., & Keller, W. (1999). An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science. 286, 1146-49.



RNA editing, adenosine, deaminase, inosine, trypanosomatids