ABSTRACTTransfer RNA (tRNA) genes are among the most highly transcribed genes in the genome due to their central role in protein synthesis. However, there is evidence for a broad range of gene expression across tRNA loci. This complexity, combined with post-transcriptional modifications and high sequence identity across transcripts, has severely limited our collective understanding of tRNA gene expression regulation and evolution. We establish correlates to tRNA gene expression and develop a

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... A gene classification method that minimally requires a single reference genome per species of interest and achieves accuracy comparable to epigenomic and ChIP-Seq assays. We observe that G/C content and CpG density surrounding tRNA loci is exceptionally well correlated with tRNA gene activity, supporting a prominent regulatory role of the local genomic context in combination with internal sequence features. We use our classifications in conjunction with a novel tRNA gene ortholog set spanning 29 placental mammals to infer that tRNA gene expression regulation evolves slowly. The success of our method has fundamental implications to tRNA annotation and variant prioritization. Its simplicity and robustness suggests facile application to other clades and timescales, as well as to explore functional diversification of members of large gene families.