Rare Codons Are Not Translational Slowdown Factors in Mammals, but Passive Byproducts of Genomic Constraints
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.
References
Hanson, G. & Coller, J. Codon optimality, bias and usage in translation and mRNA decay. Nature reviews. Molecular cell biology 19, 20-30 (2018).
Cannarozzi, G. et al. A role for codon order in translation dynamics. Cell 141, 355-367 (2010).
Ding, W. et al. Rare codon recoding for efficient noncanonical amino acid incorporation in mammalian cells. Science 384, 1134-1142 (2024).
Chamary, J. V. & Hurst, L. D. Evidence for selection on synonymous mutations affecting stability of mRNA secondary structure in mammals. Genome biology 6, R75 (2005).
Zhang, H. et al. Algorithm for optimized mRNA design improves stability and immunogenicity. Nature 621, 396-403 (2023).
Liu, Y., Yang, Q. & Zhao, F. Synonymous but Not Silent: The Codon Usage Code for Gene Expression and Protein Folding. Annual review of biochemistry 90, 375-401 (2021).
Schroder, N. W. & Schumann, R. R. Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious disease. The Lancet. Infectious diseases 5, 156-164 (2005).
Zhang, J. & Qian, W. Functional synonymous mutations and their evolutionary consequences. Nature reviews. Genetics 26, 789-804 (2025).
Ikemura, T. Correlation between the abundance of yeast transfer RNAs and the occurrence of the respective codons in protein genes. Differences in synonymous codon choice patterns of yeast and Escherichia coli with reference to the abundance of isoaccepting transfer RNAs. Journal of molecular biology 158, 573-597 (1982).
Grantham, R., Gautier, C., Gouy, M., Mercier, R. & Pave, A. Codon catalog usage and the genome hypothesis. Nucleic acids research 8, r49-r62 (1980).
Grantham, R., Gautier, C., Gouy, M., Jacobzone, M. & Mercier, R. Codon catalog usage is a genome strategy modulated for gene expressivity. Nucleic acids research 9, r43-74 (1981).
Ikemura, T. Codon usage and tRNA content in unicellular and multicellular organisms. Molecular biology and evolution 2, 13-34 (1985).
Thornlow, B. P. et al. Predicting transfer RNA gene activity from sequence and genome context. Genome research 30, 85-94 (2020).
Chan, P. P. & Lowe, T. M. tRNAscan-SE: Searching for tRNA Genes in Genomic Sequences. Methods in molecular biology 1962, 1-14 (2019).
Hershberg, R. & Petrov, D. A. Selection on codon bias. Annual review of genetics 42, 287-299 (2008).
Wu, Q. & Bazzini, A. A. Translation and mRNA Stability Control. Annual review of biochemistry 92, 227-245 (2023).
Quax, T. E., Claassens, N. J., Soll, D. & van der Oost, J. Codon Bias as a Means to Fine-Tune Gene Expression. Molecular cell 59, 149-161 (2015).
Gustafsson, C., Govindarajan, S. & Minshull, J. Codon bias and heterologous protein expression. Trends in biotechnology 22, 346-353 (2004).
Kim, C. H., Oh, Y. & Lee, T. H. Codon optimization for high-level expression of human erythropoietin (EPO) in mammalian cells. Gene 199, 293-301 (1997).
Hu, X., Shi, Q., Yang, T. & Jackowski, G. Specific replacement of consecutive AGG codons results in high-level expression of human cardiac troponin T in Escherichia coli. Protein expression and purification 7, 289-293 (1996).
Leppek, K. et al. Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. Nature communications 13, 1536 (2022).
Ramirez-Carrozzi, V. R. et al. A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling. Cell 138, 114-128 (2009).
Reik, W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447, 425-432 (2007).
Li, J., Zhou, J., Wu, Y., Yang, S. & Tian, D. GC-Content of Synonymous Codons Profoundly Influences Amino Acid Usage. G3 5, 2027-2036 (2015).
Nabiyouni, M., Prakash, A. & Fedorov, A. Vertebrate codon bias indicates a highly GC-rich ancestral genome. Gene 519, 113-119 (2013).
Novoa, E. M. & Ribas de Pouplana, L. Speeding with control: codon usage, tRNAs, and ribosomes. Trends in genetics: TIG 28, 574-581 (2012).
Lucas, M. C. et al. Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing. Nature biotechnology 42, 72-86 (2024).
Behrens, A., Rodschinka, G. & Nedialkova, D. D. High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq. Molecular cell 81, 1802-1815 e1807 (2021).
Shaner, N. C. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nature methods 10, 407-409 (2013).
Martin, S. L., Vrhovski, B. & Weiss, A. S. Total synthesis and expression in Escherichia coli of a gene encoding human tropoelastin. Gene 154, 159-166 (1995).
Wang, B. Q., Lei, L. & Burton, Z. F. Importance of codon preference for production of human RAP74 and reconstitution of the RAP30/74 complex. Protein expression and purification 5, 476-485 (1994).
Goldman, E., Rosenberg, A. H., Zubay, G. & Studier, F. W. Consecutive low-usage leucine codons block translation only when near the 5' end of a message in Escherichia coli. Journal of molecular biology 245, 467-473 (1995).
Kane, J. F. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Current opinion in biotechnology 6, 494-500 (1995).
Rosano, G. L. & Ceccarelli, E. A. Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microbial cell factories 8, 41 (2009).
Kozak, M. Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361, 13-37 (2005).
Kozak, M. Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187-208 (1999).
Prabhakar, A., Choi, J., Wang, J., Petrov, A. & Puglisi, J. D. Dynamic basis of fidelity and speed in translation: Coordinated multistep mechanisms of elongation and termination. Protein science: a publication of the Protein Society 26, 1352-1362 (2017).
Siller, E., DeZwaan, D. C., Anderson, J. F., Freeman, B. C. & Barral, J. M. Slowing bacterial translation speed enhances eukaryotic protein folding efficiency. Journal of molecular biology 396, 1310-1318 (2010).
Tuller, T. et al. An evolutionarily conserved mechanism for controlling the efficiency of protein translation. Cell 141, 344-354 (2010).
Brito Querido, J., Diaz-Lopez, I. & Ramakrishnan, V. The molecular basis of translation initiation and its regulation in eukaryotes. Nature reviews. Molecular cell biology 25, 168-186 (2024).
Sonenberg, N. & Hinnebusch, A. G. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136, 731-745 (2009).
Yu, C. H. et al. Codon Usage Influences the Local Rate of Translation Elongation to Regulate Co-translational Protein Folding. Molecular cell 59, 744-754 (2015).
Charneski, C. A. & Hurst, L. D. Positively charged residues are the major determinants of ribosomal velocity. PLoS biology 11, e1001508 (2013).
Sauna, Z. E. & Kimchi-Sarfaty, C. Understanding the contribution of synonymous mutations to human disease. Nature reviews. Genetics 12, 683-691 (2011).
Dhindsa, R. S. et al. A minimal role for synonymous variation in human disease. American journal of human genetics 109, 2105-2109 (2022).
Kruglyak, L. et al. Insufficient evidence for non-neutrality of synonymous mutations. Nature 616, E8-E9 (2023).
Shen, X., Song, S., Li, C. & Zhang, J. Synonymous mutations in representative yeast genes are mostly strongly non-neutral. Nature 606, 725-731 (2022).
Wang, Y. et al. mRNA vaccine: a potential therapeutic strategy. Molecular cancer 20, 33 (2021).
Liu, C. et al. mRNA-based cancer therapeutics. Nature reviews. Cancer 23, 526-543 (2023).
Li, Y. et al. Deep generative optimization of mRNA codon sequences for enhanced mRNA translation and therapeutic efficacy. Nature communications 16, 9957 (2025).
Morales, J. et al. A joint NCBI and EMBL-EBI transcript set for clinical genomics and research. Nature 604, 310-315 (2022).
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