Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides

Abstract

Stop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418-induced miscoding alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

eLife digest

Many genes provide a set of instructions needed to build a protein, which are read by structures called ribosomes through a process called translation. The genetic information contains a short, coded instruction called a stop codon which marks the end of the protein. When a ribosome finds a stop codon it should stop building and release the protein it has made.

Ribosomes do not always stop at stop codons. Certain chemicals can actually prevent ribosomes from detecting stop codons correctly, and aminoglycosides are drugs that have exactly this effect. Aminoglycosides can be used as antibiotics at low doses because they interfere with ribosomes in bacteria, but at higher doses they can also prevent ribosomes from detecting stop codons in human cells. When ribosomes do not stop at a stop codon this is called readthrough. There are different types of stop codons and some are naturally more effective at stopping ribosomes than others.

Wangen and Green have now examined the effect of an aminoglycoside called G418 on ribosomes in human cells grown in the laboratory. The results showed how ribosomes interacted with genetic information and revealed that certain stop codons are more affected by G418 than others. The stop codon and other genetic sequences around it affect the likelihood of readthrough. Wangen and Green also showed that sequences that encourage translation to stop are more common in the area around stop codons.

These findings highlight an evolutionary pressure driving more genes to develop strong stop codons that resist readthrough. Despite this, some are still more affected by drugs like G418 than others. Some genetic conditions, like cystic fibrosis, result from incorrect stop codons in genes. Drugs that promote readthrough specifically in these genes could be useful new treatments.