How much is Transcription-associated Mutagenesis Driving tRNA Microevolution?

Transfer RNAs (tRNAs) are among the most highly conserved and frequently transcribed genes. Recent studies have identified transcription-associated mutagenesis (TAM) as a significant contributor to sequence variation around tRNA loci. However, the extent to which TAM drives allelic variation in tRNAs remains unclear, largely due to the confounding effects of strong selection pressures to maintain their structural integrity. This complexity arises because TAM-induced mutations primarily involve nucleotide transitions, which tend to preserve base-pairing stability. To address this dichotomy at the population level, we analyzed tRNA allelic variation in contemporary Caenorhabditis elegans strains. We propose a model of tRNA microevolution driven by TAM and demonstrate that the observed secondary structure characteristics align with our predicted TAM-biased patterns. Furthermore, we developed a continuous Markov substitution model that incorporates TAM-specific mutational biases. This TAM-biased model fits the C. elegans tRNA data more effectively than standard models, such as the general time-reversible (GTR) model. Based on these results, we conclude that TAM plays a significant role in shaping tRNA allelic variation within populations. This finding is consistent with recent experimental studies on tRNA fitness in yeast, but challenges prior theoretical and computational analyses that emphasize RNA base-pairing as a primary determinant in genotype-phenotype systems.