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Hidden stop codons dictate mRNA fate by ambushing ribosomal frameshifting

ABSTRACT: 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.

ORGANISM(S): Homo sapiens Neurospora crassa

PROVIDER: GSE303542 | GEO | 2025/07/31

REPOSITORIES: GEO

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Project description:Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 allele of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.

Project description:Proline-rich antimicrobial peptide apidaecin (Api) inhibits bacterial protein synthesis in a distinctive way. In vitro biochemical and structural studies showed that Api binds in the nascent peptide exit tunnel of the ribosome that has completed translation of the gene and has released the newly-synthesized protein. Once in the tunnel, Api traps the release factors on the post-termination ribosome leading to cessation of translation. The mode of Api action in the bacterial cell, however, had remained unknow. By analyzing distribution of ribosomes on mRNAs in Api-treated cells using Ribo-seq approach we uncovered a range of effects that stem from the unique mechanism of Api action. Exposure of bacterial cells to Api results in arrest of translation at stop codons, likely in a post-release state, due to the immediate Api action, as well as in a pre-release state due to the depletion of available release factors. In addition, arrest of translation at the end of the open reading frame (ORF) leads to a pronounced queuing of the translating ribosomes behind the one arrested at the stop codon. One of the major consequences of the Api action is a dramatically increased stop codon bypass by the ribosomes paused in a pre-release state, leading to accumulation of proteins with C-terminal extensions, as confirmed by whole-cell proteomics analysis. Stop codon bypass, that occurs either in 0-frame, by misincorporation of a near-cognate aminoacyl-tRNA at the stop codon, or via frameshifting allows for a fraction of the translating ribosomes to reach the 3’ ends of mRNA transcripts. The pervasive stalling of pre-release ribosomes at the stop codons and at the RNA transcripts ends triggers activation of the cellular ribosome rescue systems which, likely due to Api action as well, remain ineffective. While major Api effects are directed towards the ribosome at the end of the ORFs, cells exposed to Api show a somewhat increased ribosome occupancy of the start codons. Understanding the unique mode of Api to inhibit translation termination in the cell can help envisioning the development of treatments for genetic diseases and research tools for genome exploration.

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Hidden stop codons dictate mRNA fate by ambushing ribosomal frameshifting

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