Abstract: In human mitochondria, genes for tRNA(Tyr) and tRNA(Cys) overlap by a single nucleotide. From polycistronic precursors, a 3'-truncated upstream tRNA(Tyr) is released, missing the overlapping position. A subsequent editing reaction restores this position. Similar mitochondrial tRNA gene overlaps exist in all metazoans, but not in organisms such as yeast or Escherichia coli. Therefore, we asked whether tRNA overlaps are processed in these organisms. Corresponding constructs were introduced and transcripts tested for processing and editing in E. coli and yeast. E. coli produces only one functional tRNA from these precursors, indicating that tRNA overlaps are incompatible with its processing pathway. In contrast, yeast processes overlapping tRNAs similar to human mitochondria, releasing a 3'-truncated upstream tRNA. This tRNA is restored in an editing-like event, although yeast does not carry a corresponding endogenous editing substrate. These findings support the hypothesis of the evolution of editing by recruitment of a pre-existing and promiscuous editing enzyme.