Monitoring translation in all reading frames downstream of weak stop codons provides mechanistic insights into the impact of nucleotide and cellular contexts

Abstract

A stop codon entering the ribosome A-site is normally decoded by release factors that induce release of the polypeptide. Certain factors influence the efficiency of the termination which is in competition with elongation in either the same (readthrough) or an alternative (frameshifting) reading frame. To gain insight into the competition between these processes, we monitored translation in parallel from all three reading frames downstream of stop codons while changing the nucleotide context of termination sites or altering cellular conditions (polyamine levels). We found that P-site codon identity can have a major impact on the termination efficiency of the OPRL1 stop signal, whereas for the OAZ1 ORF1 stop signal, the P-site codon mainly influences the reading frame of non-terminating ribosomes. Changes to polyamine levels predominantly influence the termination efficiency of the OAZ1 ORF1 stop signal. In contrast, increasing polyamine levels stimulate readthrough of the OPRL1 stop signal by enhancing near-cognate decoding rather than by decreasing termination efficiency. Thus, by monitoring the four competing processes occurring at stop codons we were able to determine which is the most significantly affected upon perturbation. This approach may be useful for the interrogation of other recoding phenomena where alternative decoding processes compete with standard decoding.

Systematic analysis of nonsense variants uncovers peptide release rate as a novel modifier of nonsense-mediated mRNA decay efficiency

“…A previous study showed that Gly acts as a C-terminal degron and is rarely found at the C-terminal end of the normal proteome, suggesting one possible biological basis for Gly-PTC enrichment (28,29). It is also possible that Gly-PTC stimulates PTC readthrough, leading to NMD escape and some functionality of the protein product (30), or that Gly-PTC enhances NMD and elimination of the mRNA transcript.…”

“…A previous study showed that Gly acts as a C-terminal degron and is rarely found at the C-terminal end of the normal proteome, suggesting that Gly-PTC enrichment in non-essential genes may be due to efficient Gly-end truncated protein degradation (Koren et al, 2018; Lin et al, 2018). It is also possible that Gly-PTC stimulates PTC readthrough, leading to NMD escape and some functionality of the protein product (Loughran et al, 2023), or that Gly-PTC enhances NMD and elimination of the mRNA transcript. Readthrough would confer a benefit to the organism as it would preserve gene function, while elimination of the truncated protein or encoding transcript might have a neutral effect if the gene is otherwise non-essential.…”

Systematic and quantitative analysis of stop codon readthrough in Rett syndrome nonsense mutations

“…Both bear UGA stop codons which are generally most prone to TR [ 25 – 27 ]. The MeCP2 p.R198X variant further contains a cytosine in the + 4 SCC position which was previously shown to additionally increase the TR efficiency [ 26 , 45 ]. Further, p.R198X was the only variant that expressed the full-length MeCP2 isoform confirming the high basal TR of the UGA C context.…”

Mechanisms and Delivery of tRNA Therapeutics

“…Beyond the +4 position, the sequence influence seems to diminish but some effect is still exerted by nucleotides as far out as +9 . While no common consensus sequence has emerged, there is some evidence of eukaryotic sequences that bias NTC readthrough, e.g., UGA-CUAG in Drosophila , or various sequences that support viral readthrough and may enhance aminoglycoside activity , The impact of the 5′ sequence context is less well established but some studies have shown that an adenine at the −1 position may improve readthrough rates. − …”