Modification of the universally unmodified uridine-33 in a mitochondria-imported edited tRNA and the role of the anticodon arm structure on editing efficiency

Editing of tRNA has a wide phylogenetic distribution among eukaryotes and in some cases serves to expand the decoding capacity of the target tRNA. We previously described C-to-U editing of the wobble position of the Imported tRNA(Trp) in Leishmania mitochondria, which is essential for decoding UGA codons as tryptophan. Here we show the complete set of nucleotide modifications In the anticodon arm of the mitochondrial and cytosolic tRNA(Trp) as determined by electrospray ionization mass spectrometry. This analysis revealed extensive mitochondria-specific post-transcriptional modifications, including the first example of thiolation of U33, the "universally unmodified" uridine. In light of the known rigidity Imparted on sugar conformation by thiolation, our discovery of a thiolated U33 suggests that conformational flexibility is not a universal feature of the anticodon structural signature. In addition, the in vivo analysis of tRNA(Trp) variants presented shows a single base-pair reversal in the anticodon stem of tRNA(Trp) is sufficient to abrogate editing in vivo, indicating that subtle changes in anticodon structure can have drastic effects on editing efficiency.