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: two overlapping tRNAs produced from opposite strands of the same locus. These mirror tRNA pairs eliminate the need to retain separate loci for all tRNA genes, as a single locus can produce tRNAs to decode two different amino acids. We show that these mirror tRNAs are aminoacylated and demonstrate their presence in mitoribosomes. Furthermore, mirror tRNAs display strand-specific patterns of nucleotide modification and RNA editing, reflecting specific post-transcriptional maturation that depends on transcriptional orientation. To our knowledge, this demonstration of functional, bidirectional tRNA expression is a first for any genome or organism and reveals an unexpected strategy by which mitochondrial genomes maintain a complete set of tRNAs in the face of unrelenting gene loss. The discovery of mirror tRNAs has broad implications for the evolution of tRNA-interacting enzymes, mitochondrial biology, and even the origins of the protein synthesis machinery itself. Significance StatementMitochondrial genomes have been reduced to only a handful of genes over the roughly two billion years since the origin of the organelle. Despite this extreme gene loss, bilaterian animal mitochondrial genomes still retain a complete, minimal set of tRNAs necessary for protein synthesis. Here, we identify a previously unrecognized strategy in animal mitochondria to maintain a complete set of tRNAs - the bidirectional expression of a single genomic locus to produce two different tRNAs. These "mirror" tRNAs are aminoacylated, incorporated into mitoribosomes, and exhibit strand-specific patterns of editing and modification. This surprising coding strategy reveals how mitochondrial genomes can preserve a complete set of tRNAs despite unrelenting gene loss.