With the rapid increase of sequenced metazoan mitochondrial genomes, a detailed manual annotation is becoming more and more infeasible. While it is easy to identify the approximate location of protein-coding genes within mitogenomes, the peculiar processing of mitochondrial transcripts, however, makes the determination of precise gene boundaries a surprisingly difficult problem. We have analyzed the properties of annotated start and stop codon positions in detail, and use the inferred patterns to devise a new method for predicting gene boundaries in de novo annotations. Our method benefits from empirically observed prevalances of start/stop codons and gene lengths, and considers the dependence of these features on variations of genetic codes. Albeit not being perfect, our new approach yields a drastic improvement in the accuracy of gene boundaries and upgrades the mitochondrial genome annotation server MITOS to an even more sophisticated tool for fully automatic annotation of metazoan mitochondrial genomes.

Remolding of tRNAs is a well-documented process in mitochondrial genomes that changes the identity of a tRNA. It involves a duplication of a tRNA gene, a mutation that changes the anticodon and the loss of the ancestral tRNA gene. The net effect is a functional tRNA that is more closely related to tRNAs of a different alloacceptor family than to tRNAs with the same anticodon in related species. Beyond being of interest for understanding mitochondrial tRNA function and evolution, tRNA remolding events can lead to artifacts in the annotation of mitogenomes and thus in studies of mitogenomic evolution. Therefore, it is important to identify and catalog these events. Here we describe novel methods to detect tRNA remolding in large-scale data sets and apply them to survey tRNA remolding throughout animal evolution. We identify several novel remolding events in addition to the ones previously mentioned in the literature. A detailed analysis of these remoldings showed that many of them are derived from ancestral events.