Synonymous codon usage regulates translation initiation

Nonoptimal synonymous codons repress gene expression, but the underlying mechanisms are poorly understood. We and others have previously shown that nonoptimal codons slow translation elongation speeds and thereby trigger messenger RNA (mRNA) degradation. Nevertheless, transcript levels are often insufficient to explain protein levels, suggesting additional mechanisms by which codon usage regulates gene expression. Using reporters in human and Drosophila cells, we find that transcript levels account for less than half of the variation in protein abundance due to codon usage. This discrepancy is explained by translational differences whereby nonoptimal codons repress translation initiation. Nonoptimal transcripts are also less bound by the translation initiation factors eIF4E and eIF4G1, providing a mechanistic explanation for their reduced initiation rates. Importantly, translational repression can occur without mRNA decay and deadenylation, and it does not depend on the known nonoptimality sensor, CNOT3. Our results reveal a potent mechanism of regulation by codon usage where nonoptimal codons repress further rounds of translation. [Display omitted] •Synonymous codon usage impacts protein expression beyond mRNA levels•Nonoptimal transcripts are engaged by fewer ribosomes•Reduced eIF4E binding is required to repress translation initiation•Translational repression is not a consequence of mRNA decay nor deadenylation Barrington et al. demonstrate that nonoptimal codon usage reduces protein expression post-transcriptionally by repressing translation initiation. These findings reconcile a long-standing contradiction in the field between slow elongation speed and reduced ribosome occupancy and suggest that elongation and initiation machinery cooperate to regulate gene expression.