# Less is more: slow-codon windows enhance eGFP mRNA resilience against RNA interference

## TL;DR

This study shows that using slow-codons in mRNA can make it more resilient to RNA interference, affecting how long it lasts in human cells.

## Contribution

The study reveals that slow-codon windows enhance mRNA resilience against RNA interference, a previously underexplored aspect of codon choice.

## Key findings

- mRNAs with slow-codon windows have shorter lifetimes under normal conditions.
- Slow-codon mRNAs show similar stability in the presence of siRNA as unmutated mRNAs.
- Ribosome density correlates with mRNA stability in a cell-type- and codon-position-specific manner.

## Abstract

Extensive efforts have been devoted to enhancing the translation efficiency of mRNA delivered to mammalian cells via codon optimization. However, the impact of codon choice on mRNA stability remains underexplored. In this study, we investigated the influence of codon usage on mRNA degradation kinetics in cultured human cell lines using live-cell imaging on single-cell arrays. By measuring mRNA lifetimes at the single-cell level for synthetic mRNA constructs, we confirmed that mRNAs containing slowly translated codon windows have shorter lifetimes. Unexpectedly, these mRNAs did not exhibit decreased stability in the presence of small interfering RNA (siRNA) compared with the unmutated sequence, suggesting an interference of different concurrent degradation mechanisms. We employed stochastic simulations to predict ribosome density along the open reading frame, revealing that the ribosome densities correlated with mRNA stability in a cell-type- and codon-position-specific manner. In summary, our results suggest that the effect of codon choice and its influence on mRNA lifetime is context-dependent with respect to cell type, codon position and RNA interference.