Differential requirements for mRNA folding partially explain why highly expressed proteins evolve slowly

The cause of the tremendous among-protein variation in the rate of sequence evolution is a central subject of molecular evolution. Expression level has been identified as a leading determinant of this variation among genes encoded in the same genome, but the underlying mechanisms are not fully understood. We here propose and demonstrate that a requirement for stronger folding of more abundant mRNAs results in slower evolution of more highly expressed genes and proteins. Specifically, we show that: (i) the higher the expression level of a gene, the greater the selective pressure for its mRNA to fold; (ii) random mutations are more likely to decrease mRNA folding when occurring in highly expressed genes than in lowly expressed genes; and (iii) amino acid substitution rate is negatively correlated with mRNA folding strength, with or without the control of expression level. Furthermore, synonymous (d(S)) and nonsynonymous (d(N)) nucleotide substitution rates are both negatively correlated with mRNA folding strength. However, counterintuitively, d(S) and d(N) are differentially constrained by selection for mRNA folding, resulting in a significant correlation between mRNA folding strength and d(N)/d(S), even when gene expression level is controlled. The direction and magnitude of this correlation is determined primarily by the G+C frequency at third codon positions. Together, these findings explain why highly expressed genes evolve slowly, demonstrate a major role of natural selection at the mRNA level in constraining protein evolution, and reveal a previously unrecognized and unexpected form of nonprotein-level selection that impacts d(N)/d(S).