Rare Codons as Passive Byproducts of Genomic Constraints, Not Translational Regulators in Mammals
Abstract
The prevailing view holds that rare codons function as evolutionarily conserved modulators of translation kinetics, a concept that has provided a critical framework for interpreting synonymous mutations in disease and guiding coding sequence optimization in gene therapy. Our findings, however, challenge this premise by demonstrating that rare codons are primarily passive byproducts of genome stability constraints, rather than adaptive regulators of translation. Supporting this, genomic analyses reveal that rare codons, which are predominantly CpG- or TpA-ending, are genomically scarce; for more than half of them, the corresponding nucleotide triplet appears in the genome at a frequency that is approximately half of its occurrence in coding sequences, indicating that codon rarity reflects genome-wide sequence constraints rather than adaptive pressures for translational control. Furthermore, interrogation of tRNA abundance, via both genomic copy number and direct cellular quantification, shows no consistent correlation between codon rarity and cognate tRNA scarcity. Crucially, direct experimental assessment via cytoplasmic microinjection of engineered mRNAs into mouse embryos demonstrates that even a substantial artificial increase in rare codon load has a negligible impact on protein synthesis kinetics. Consequently, we conclude that rare codons cannot be regarded as an independent factor limiting translation rates in mammalian cells. This insight demands a reassessment of the role of synonymous mutations in disease and expands the design principles for mRNA-based therapeutics by alleviating the presumed constraint of rare codon avoidance.
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The authors declare no competing interests to disclose.
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