Attenuating ribosome load improves protein output from mRNA by limiting translation-dependent mRNA decay

Developing an effective mRNA therapeutic often requires maximizing protein output per delivered mRNA molecule. We previously found that coding sequence (CDS) design can substantially affect protein output, with mRNA variants containing more optimal codons and higher secondary structure yielding the highest protein outputs due to their slow rates of mRNA decay. Here, we demonstrate that CDS-dependent differences in translation initiation and elongation rates lead to differences in translation- and deadenylation-dependent mRNA decay rates, thus explaining the effect of CDS on mRNA half-life. Surprisingly, the most stable and highest-expressing mRNAs in our test set have modest initiation/elongation rates and ribosome loads, leading to minimal translation-dependent mRNA decay. These findings are of potential interest for optimization of protein output from therapeutic mRNAs, which may be achieved by attenuating rather than maximizing ribosome load. [Display omitted] •Exogenously delivered mRNAs undergo translation-dependent decay through deadenylation•Nonoptimal codons lead to slow elongation, high ribosome load, and fast mRNA decay•mRNAs with fast initiation rates have high ribosome loads and fast mRNA decay•The most stable mRNAs are those with optimal codons and moderate ribosome loads Bicknell et al. demonstrate that exogenously delivered mRNAs are degraded in a translation-dependent manner at a rate that correlates with ribosome load. ORF sequences leading to high ribosome load cause faster translation-dependent decay. The most stable mRNA sequences are those with the fewest ribosomes due to slower rates of translation initiation.