Establishment of the early genetic code likely required strategies to ensure translational accuracy and inevitably involved tRNA post-transcriptional modifications. One such modification, wybutosine/wyosine is crucial for translational fidelity in Archaea and Eukarya; yet it does not occur in Bacteria and has never been described in mitochondria. Here, we present genetic, molecular and mass spectromery data demonstrating the first example of wyosine in mitochondria, a situation thus far unique to kinetoplastids. We also show that these modifications are important for mitochondrial function, underscoring their biological significance. This work focuses on TyW1, the enzyme required for the most critical step of wyosine biosynthesis. Based on molecular phylogeny, we suggest that the kinetoplastids pathways evolved via gene duplication and acquisition of an FMN-binding domain now prevalent in TyW1 of most eukaryotes. These findings are discussed in the context of the extensive U-insertion RNA editing in trypanosome mitochondria, which may have provided selective pressure for maintenance of mitochondrial wyosine in this lineage.

Department of Microbiology and The Center for RNA Biology The Ohio State University Columbus OH 43210 USA

Department of Microbiology and The Center for RNA Biology The Ohio State University Columbus OH 43210 USA Rieveschl Laboratories for Mass Spectrometry Department of Chemistry University of Cincinnati Cincinnati OH 45221 USA Ohio State Biochemistry Program The Ohio State University Columbus Ohio 43210 USA

Institute of Parasitology Biology Centre and Faculty of Sciences University of South Bohemia 37005 České Budějovice Czech Republic

Institute of Parasitology Biology Centre and Faculty of Sciences University of South Bohemia 37005 České Budějovice Czech Republic Canadian Institute For Advanced Research Toronto ON M5G 1Z8 Canada

Rieveschl Laboratories for Mass Spectrometry Department of Chemistry University of Cincinnati Cincinnati OH 45221 USA

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How the intracellular partitioning of tRNA and tRNA modification enzymes affects mitochondrial function

Retrograde nuclear transport from the cytoplasm is required for tRNATyr maturation in T. brucei

Editing and methylation at a single site by functionally interdependent activities