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).
Highlights
To complete synthesis of a mature protein, ribosomes must terminate translation accurately at the end of each coding sequence
Normal protein synthesis was measured by the expression of full-length FLuc, and Stop codon readthrough (SCR) was measured by the expression of Nano Luciferase (NLuc) when a premature termination codon (PTC) was inserted at position R154X (UGA)
While expressing each luciferase from a separate mRNA can introduce variability in RNA levels between messages, this strategy avoids many of the pitfalls that can confound interpretation of bicistronic reporters for studying stop codon readthrough (Loughran et al, 2017; Terenin et al, 2017)
Summary
To complete synthesis of a mature protein, ribosomes must terminate translation accurately at the end of each coding sequence. Following stop codon recognition by release factors, the nascent peptide is hydrolyzed by eRF1 releasing the mature protein product (Frolova et al, 1999; Zhouravleva et al, 1995) and ribosomes are subsequently removed from the mRNA by recycling (Barthelme et al, 2011; Pisarev et al, 2010; Shoemaker and Green, 2011). Discrimination of normal and problematic termination contexts must occur independently of the nucleotide sequence of the stop codon since identical stop codons (UAA, UAG, and UGA) signal translation termination at both NTCs and PTCs. In mammals, a strong signal for NMD derives from the position of the stop codon relative to that of a protein complex known as the Exon-Junction-Complex (EJC) deposited upstream of each splice junction during splicing of the mRNA
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