Hidden stop codons dictate mRNA fate by ambushing ribosomal frameshifting

In eukaryotes, the intrinsic mRNA stability is generally dictated by codon usage bias, the uneven preferences for synonymous codons, in a translation-dependent manner. However, the conserved mechanism underlining this phenomenon remains elusive. Hidden stop codons (HSCs), which are stop codons located in the +1 or -1 frame relative to canonical open reading frames (ORF), are sidespread across all genomes but have long been overlooked. We demonstrate that in both fungi and mammals, HSCs play an important role in regulating mRNA decay across the genome by immediately terminating out-of-frame translations promoted by nonoptimal codons, primarily through +1 ribosomal frameshifting. In the filamentous fungus Neurospora, partially deleting HSCs via synonymous substitutions in the clock gene frequency disrupts the circadian rhythm due to increased mRNA stability. Similarly, in human cells, impaired translation termination caused by rapid depletion of eRF1 leads to globally increased stability of mRNAs enriched with non-optimal codons and HSCs, which are in part degraded through the NMD pathway via UPF1. Given that from yeasts to humans the occurrence of HSCs is genome-widely associated with the presence of nonoptimal codons, these findings suggest that in eukaryotes, HSCs might coevolve with codon usage to modulate mRNA stabilities via immediately terminating different levels of ribosomal frameshifting events promoted by nonoptimal codons. SLAM-seq assay was performed for HCT116 cell line and N. crassa strains ( WT and upf1 KO) to observe mRNA decay profile. Timepoints: 0, 10, 20, 40, 60 minutes. Ribo-seq assay was performed for WT and eRF1 KD HCT116 cell line