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Functional synonymous mutations and their evolutionary consequences

Nature Reviews Genetics volume 26, pages 789–804 (2025)Cite this article

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Abstract

Synonymous mutations are coding mutations that do not alter protein sequences. Commonly thought to have little to no functional consequence, synonymous mutations have been widely used in evolutionary analyses that require neutral markers, including those foundational for the neutral theory. However, recent studies suggest that synonymous mutations can influence nearly every step in the expression of genetic information and may often be strongly non-neutral. We review the extent and mechanisms of these phenotypic and fitness effects and discuss the implications of the functionality and non-neutrality of synonymous mutations for various analyses and conclusions pertinent to genetics, evolution, conservation and disease.

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Fig. 1: Multifaceted effects of synonymous mutations on the expression of genetic information in eukaryotes.
Fig. 2: Various patterns of codon usage bias and their causes.
Fig. 3: Fitness effects of synonymous and nonsynonymous mutations or polymorphisms in four systematic studies.

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Acknowledgements

The authors thank S. Song, S. Zhang and J. Kong for technical assistance, and members of our laboratories for valuable comments. J.Z. was supported by the U.S. National Institutes of Health (R35GM139484) and W.Q. was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA28030402), the Biological Breeding-National Science and Technology Major Project (2023ZD04073) and Guangdong Provincial Key Laboratory of Synthetic Genomics (2023B1212060054).

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Authors and Affiliations

  1. Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI, USA

    Jianzhi Zhang

  2. State Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Wenfeng Qian

  3. University of Chinese Academy of Sciences, Beijing, China

    Wenfeng Qian

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  1. Jianzhi Zhang
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  2. Wenfeng Qian
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Correspondence to Jianzhi Zhang or Wenfeng Qian.

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Glossary

A-to-I editing

Enzymatic alteration of RNA molecules consisting of the conversion of adenosines (A) to inosines (I) at specific positions.

Clonal interference

Competition among genotypes with different beneficial mutations in an asexual population.

Coalescent theory

A mathematical theory of population genetics that traces all alleles of a gene sampled from a population to a single ancestral copy.

Codon usage bias

(CUB). The phenomenon in which synonymous codons of an amino acid are unequally used in a genome.

Cycloheximide

A fungicide often used to block eukaryotic translational elongation in experiments.

Deep mutational scanning

An experimental approach for measuring the effects of individual nucleotides in a DNA segment by creating many mutants of the DNA followed by high-throughput functional/fitness assays of the mutants.

Effective population size

(Ne). Number of individuals in an ideal (that is, Wright–Fisher) population that results in the same amount of genetic drift as in the actual population considered.

Fitness

A quantitative representation of the ability of an individual to pass its genome to the next generation.

Genome-wide association studies

Investigations of genome-wide sets of genetic variants in groups of individuals to find variants associated with traits of interest.

Kinetic proofreading

Mechanism that allows enzymes, particularly those involved in DNA replication, RNA transcription and protein synthesis, to enhance their fidelity by discriminating between correct and incorrect substrates. The accuracy of this process is higher than expected solely based on the difference in activation energy between forming correct products and incorrect products.

Linkage disequilibrium

Non-random association of alleles of different loci in a population.

m6A methylation

Methylation at the nitrogen-6 position of adenosines at specific positions in an RNA molecule.

MicroRNA

Single-stranded, non-coding RNA molecules of 21 to 23 nucleotides that bind to mRNAs to cause mRNA degradation or suppress mRNA translation.

Modern synthesis

Prevailing evolutionary theory developed in the 1930s to the 1940s by combining Darwin’s theory of evolution by natural selection with a population-oriented view of Mendelian genetics.

mRNA folding strength

Reduction in free energy of a folded mRNA molecule relative to its unfolded form.

Mutation accumulation

A genetic experiment in which a population of organisms is propagated through repeated population bottlenecks to minimize the impact of natural selection.

Mutation bias

A phenomenon in which some types of mutation occur more frequently than other types.

Near-cognate tRNAs

tRNAs carrying an amino acid that differs from that encoded by the codon of concern and whose anticodon does not pair with the codon at exactly one nucleotide position.

Neutral mutations

Genetic changes having selection coefficients that are smaller than the inverse of the effective population size.

Neutral theory

An evolutionary theory positing that most interspecific differences and intraspecific polymorphisms at the DNA or protein sequence level are selectively neutral rather than adaptive.

Nonsynonymous mutations

Point mutations in a coding sequence that alter the encoded protein sequence.

Nonsynonymous substitution rate

(dN). Number of nonsynonymous nucleotide substitutions per nonsynonymous site between two homologous coding sequences.

Nucleosome

The basic structural unit of DNA packaging in eukaryotes consisting of a segment of DNA wound around eight histone proteins.

Polyadenylation

Part of the process of mRNA maturation in which a nascent RNA transcript is cleaved at a particular site and subsequently becomes the object of the addition of a poly-A tail.

Preferred codons

Codons that are used more frequently than the average of all codons of the same amino acid in highly expressed genes of a genome.

Protospacer adjacent motif

A short DNA sequence of usually 2–6 nucleotides required for a Cas nuclease in the CRISPR system to cut; it is generally found 3–4 nucleotides downstream from the cut site.

Pseudogene

A nonfunctional segment of DNA that is derived from a previously functional gene.

Quantitative trait locus mapping

A method to determine the chromosomal regions or genetic variants affecting the variation of a quantitative trait among individuals of a species.

Ribosome E-site

5’-most of the three tRNA binding sites in a ribosome that allows a deacylated tRNA to exit. The 3’-most of the three tRNA binding sites in a ribosome that selects charged tRNA molecules during protein synthesis is known as A-site.

Ribosome P-site

Middle of the three tRNA binding sites in a ribosome that holds the tRNA linked to the nascent polypeptide chain.

Selection coefficient

(s). The difference in relative fitness between a mutant and the wild type.

Shine–Dalgarno sequence

Sequence motif in mRNA that recruits ribosomes through interaction with the anti-Shine–Dalgarno sequence in 16S ribosomal RNA of prokaryotes.

Site frequency spectrum

Distribution of the allele frequencies of a set of loci such as single-nucleotide polymorphisms in a population or sample of individuals.

Splicing

A step in pre-mRNA processing that removes introns and joins exons to form mature mRNAs.

Synonymous mutations

Point mutations in a coding sequence that do not alter the encoded protein sequence.

Synonymous substitution rate

(dS). Number of synonymous nucleotide substitutions per synonymous site between two homologous coding sequences.

Unpreferred codons

Codons that are used less frequently than the average of all codons of the same amino acid in highly expressed genes of a genome.

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Zhang, J., Qian, W. Functional synonymous mutations and their evolutionary consequences. Nat Rev Genet 26, 789–804 (2025). https://doi.org/10.1038/s41576-025-00850-1

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