Expression of four mitochondrial tRNAs from only two loci

Transfer RNAs (tRNAs) are among the few genes retained in animal mitochondrial genomes after more than a billion years of gene loss. These ancient bacterial vestiges are often structurally aberrant and less stable than their bacterial or cytosolic tRNA counterparts. In some lineages, mitochondrial tRNAs (mt-tRNAs) have become so truncated that the loss of one or both arms has expanded our understanding of what constitutes a functional tRNA. Here, we report another radical departure from canonical tRNA gene architecture in animal mitochondria: two tRNA loci that produce overlapping tRNAs from both strands. These ‘mirror’ tRNA pairs eliminate the need to retain separate loci for all tRNA genes, as a single locus can produce two tRNAs that decode two different amino acids. We show that these mirror tRNAs are aminoacylated and present on mitoribosomes. Furthermore, mirror tRNAs display strand-specific patterns of nucleotide modification and RNA editing, reflecting specific post-transcriptional maturation that depends on transcriptional orientation. This bidirectional expression mechanism reveals an unexpected strategy by which mitochondrial genomes maintain a complete set of tRNAs in the face of unrelenting gene loss.