Mapping the Inter- and Intra-genic Codon Usage Landscape in Homo sapiens

Although the genetic code is degenerate, codon selection is non-random and reflects significant functional constraints. Codon usage bias (CUB) acts as a layer of post-transcriptional regulation, influencing mRNA stability, translation kinetics, and co-translational protein folding. While CUB is well-characterized in unicellular organisms, its regulatory scope and functional consequences in humans remain complex and less defined. Our study offers a comprehensive evaluation of human codon usage. We report that genes exhibiting the strongest codon bias are enriched in high-stoichiometry biological processes, such as skin development and oxygen/carbon dioxide transport, and harbor significantly fewer synonymous variants than expected (ρ = -0.24, p < 2.2 x 10 ^-16 ). Furthermore, we find that codon optimization is spatially regulated: it is significantly more pronounced in structured protein domains compared to intrinsically disordered regions (IDRs) (Cliff’s Δ= 0.26, p < 2.2 x 10 ^-16 ). Consistent with translational selection, the most frequently used codons are supported by higher tRNA gene copy numbers (ρ = 0.49, p < 6.4 x 10 ^-4 ). Finally, by correcting for GC3 content, we reveal that the apparent correlation between ENC and adaptation indices (CAI/tAI) vanishes, allowing us to disentangle mutational pressure from translational selection. Collectively, our findings position codon usage bias as a central, evolutionarily conserved regulator of translation and protein folding in humans. Our results provide a comprehensive and integrated view of intergenic and intragenic codon usage bias in humans, reinforcing the biological relevance of synonymous codon choice in shaping translational dynamics and protein biogenesis. This provides a refined framework for interpreting synonymous variation and guiding functional genomics.