By Gary Loughran, Head of Biology
Introduction
Mutations that introduce a premature termination codon (PTC) into the mRNA lead to the synthesis of truncated and usually non-functional proteins that may cause disease. Furthermore, mRNA surveillance pathways, such as the nonsense-mediated mRNA decay (NMD) pathway, can compound the impact of PTCs by degrading mutant mRNAs to prevent the accumulation of potentially hazardous truncated proteins. PTC therapeutic approaches aim to induce ribosomes to selectively read through PTCs, allowing the production of some full-length functional proteins as well as restoring mRNA levels. Assessing preferential readthrough on PTCs over normal stop codons is critical for PTC therapeutic approaches. Ribosome profiling, a cutting-edge technique that provides a high-resolution snapshot of ribosome positions on mRNAs, is an invaluable tool for assessing the efficacy and impact of PTC therapeutic approaches transcriptome-wide. This blog explores the methodology, applications, and implications of using ribosome profiling to evaluate PTC therapeutic approaches.
Understanding Translation Termination and Stop Codon Readthrough
Translation termination involves the hydrolytic release of the nascent polypeptide chain from the peptidyl-tRNA by protein release factors upon encountering a stop codon (UAA, UAG or UGA in the standard genetic code) in the ribosome A-site. In most eukaryotes, a single release factor (eRF1) recognizes all three stop codons. Normally the efficiency of termination is very high (>99.9%) but is influenced by cis-acting elements (Brown et al., 1990; Skuzeski et al., 1991; Namy et al., 2001; Loughran et al., 2014) and/or trans-acting factors (Beznosková et al., 2015; Loughran et al., 2023). Reduced termination efficiency means a decrease in the frequency of stop codon recognition by eRF1 and its subsequent and transient departure from the ribosome. This, in turn, allows increased sampling of the stop codon by charged tRNAs, including near-cognate tRNAs that can occasionally decode stop codons and permit continued translation until the next stop codon is encountered by the elongating ribosome.
Some readthrough-inducing compounds, such as the aminoglycoside antibiotics, can reduce the ribosome’s proof-reading ability and facilitate near-cognate decoding of stop codons. Although some aminoglycosides showed promise in lab-based experiments, in the clinic, severe drug toxicity at therapeutically relevant concentrations limits their long-term utility. Lower concentrations in combination with compounds that can reduce termination efficiency may be a solution to the toxicity issue. Recently, compounds that lower eRF1 levels have shown PTC readthrough promise when combined with aminoglycosides (Sharma et al., 2021; Gurzeler et al., 2023). With the successes of RNA delivery methods, encouraging strategies using engineered tRNAs fully cognate to one of the stop codons are emerging (Lueck et al., 2019; Albers et al., 2023)
Since most PTC readthrough approaches target general components of the translation apparatus or the stop codons themselves (tRNA-based therapies), there is potential to induce indiscriminate or promiscuous readthrough at normal stop codons as well as PTCs. To identify possible off-target effects of PTC therapeutic strategies it is important to determine the extent of readthrough globally. Ribosome profiling is an ideal approach to check for a general increase in stop codon readthrough transcriptome-wide.
How do PTC therapeutic approaches selectively target PTCs instead of normal stop codons?
Several reports describing PTC readthrough suggest more efficient readthrough of PTCs compared to normal stop codons which implies differences in termination efficiency (Sharma et al., 2021; Albers et al., 2023). What are the main differences between PTCs and normal stop codons that could allow selectivity? The most obvious difference is positional. PTCs are within the protein coding sequence whereas normal stop codons are, by definition, at the ends of protein coding sequences. It has been proposed that poly-A binding protein enhances interactions of the termination complex with the ribosome to increase termination efficiency (Ivanov et al., 2016). Since poly-A tails may be closer to normal stop codons than PTCs, this could provide a possible rationale for lower termination efficiency at PTCs.
Another difference is that normal stop codons have evolved strong termination contexts to prevent readthrough that could result in the synthesis of undesired C-terminally extended proteoforms. On the other hand, PTCs are generated by random mutation and are more likely than normal stop codons to be found within less favourable termination contexts.
It may be possible to exploit these position-specific and context-specific differences for PTC therapeutic approaches, however, one consideration is that not all normal stop codons are located at the ends of protein coding sequences. In higher eukaryotes many mRNAs (most mRNAs in some species like human) contain an upstream open reading frame (uORF) in their 5′ leader (or 5′UTR). Of course uORFs have stop codons and, in terms of their position within a mRNA, they can be viewed as PTCs. Readthrough of uORF stop codons could alter global translation – especially where the extended uORF overlaps with the main protein coding ORF. Furthermore, readthrough of uORFs could potentially lead to the synthesis and presentation of peptides to T cells, and elicit off-target cellular immune responses Ribosome profiling can shed light on these possibilities since undesirable uORF readthrough upon PTC treatment may induce global changes to the translatome.
Ribosome profiling can help assess the impact of PTC therapeutic approaches
Ribosome profiling involves sequencing the regions of mRNAs that are within ribosomes at the time of the experiment, providing a high-resolution snapshot of ribosome positions across the transcriptome (Ingolia et al., 2009). This technique allows researchers to identify not only which regions of mRNAs are being actively translated into proteins but also, due to the sub-codon resolution afforded by ribosome profiling, to infer which reading frame is being translated. This makes ribosome profiling an especially powerful tool for assessing the transcriptome-wide impact of strategies that target the general translation apparatus such as some PTC therapeutic approaches. Not only can ribosome profiling directly detect and quantify readthrough by identifying ribosome footprints downstream of PTCs, it can also infer off-target effects that alter translation globally.
A comparison of ribosome footprint density before and after all terminal stop codons between treated and untreated cells or tissues could highlight off-target readthrough. This comparison can be done both at the level of individual transcripts or at the global level using a metagene profile approach. This metagene profile is an average profile across multiple transcripts fixed around the stop codon. Since most normal stop codons have been selected to terminate efficiently, especially from highly translated mRNAs like house-keeping genes, then off-target readthrough upon treatment may be too subtle to detect confidently at the metagene level. The examination of ribosome profiles of those individual mRNAs with known weak termination codons may provide a more reliable measure of promiscuous readthrough.
Possible global translational changes resulting from readthrough of uORF stop codons could be assessed by standard differential gene expression analysis of ribosome profiling data. In addition to abundant uORF translation, ribosome profiling has uncovered translation of ORFs within coding regions (nested ORFs). The same possible off-target issues could arise should readthrough of nested ORFs increase. Many uORFs and internal ORFs have likely been selected for regulatory purposes without triggering the NMD pathway. Extending these ORFs could alter their resistance to NMD, since stop codon position relative to exon/exon junctions is an important trigger of NMD. Potential NMD could be detected by RNAseq which is an integral control required for ribosome profiling experiments.
Conclusion
Ribosome profiling is a powerful tool for assessing the transcriptome-wide impact of PTC therapeutic approaches. By providing high-resolution data on ribosome positions and translation dynamics, ribosome profiling enables researchers to directly observe readthrough events, measure global translation changes, and evaluate mRNA stability. The insights gained from ribosome profiling studies can guide the development of more effective PTC readthrough strategies and help minimize off-target effects. As the technology continues to advance, ribosome profiling will play an increasingly critical role in the quest to treat genetic disorders caused by nonsense mutations.
Reference
1. Albers, S., Allen, E. C., Bharti, N., et al. (2023). Engineered tRNAs suppress nonsense mutations in cells and in vivo. Nature, 618, 842–848. https://doi.org/10.1038/s41586-023-06133-1
2. Beznosková, P., Wagner, S., Jansen, M. E., von der Haar, T., & Valášek, L. S. (2015). Translation initiation factor eIF3 promotes programmed stop codon readthrough. Nucleic Acids Research, 43(10), 5099–5111. https://doi.org/10.1093/nar/gkv421
3. Brown, C. M., Stockwell, P. A., Trotman, C. N. A., & Tate, W. P. (1990). Sequence analysis suggests that tetra-nucleotides signal the termination of protein synthesis in eukaryotes. Nucleic Acids Research, 18(21), 6339-6345. https://doi.org/10.1093/nar/18.21.6339
4. Gurzeler, L. A., Link, M., Ibig, Y., Schmidt, I., Galuba, O., Schoenbett, J., Gasser-Didierlaurant, C., Parker, C. N., Mao, X., Bitsch, F., Schirle, M., Couttet, P., Sigoillot, F., Ziegelmüller, J., Uldry, A. C., Teodorowicz, W., Schmiedeberg, N., Mühlemann, O., & Reinhardt, J. (2023). Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control. Cell Reports, 42(9), 113056. https://doi.org/10.1016/j.celrep.2023.113056
5. Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., & Weissman, J. S. (2009). Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science, 324(5924), 218-223. https://doi.org/10.1126/science.1168978
6. Ivanov, A., Mikhailova, T., Eliseev, B., Yeramala, L., Sokolova, E., Susorov, D., Shuvalov, A., Schaffitzel, C., & Alkalaeva, E. (2016). PABP enhances release factor recruitment and stop codon recognition during translation termination. Nucleic Acids Research, 44(16), 7766–7776. https://doi.org/10.1093/nar/gkw635
7. Loughran, G., Chou, M. Y., Ivanov, I. P., Jungreis, I., Kellis, M., Kiran, A. M., Baranov, P. V., & Atkins, J. F. (2014). Evidence of efficient stop codon readthrough in four mammalian genes. Nucleic Acids Research, 42(14), 8928–8938. https://doi.org/10.1093/nar/gku608
8. Loughran, G., Li, X., O’Loughlin, S., Atkins, J. F., & Baranov, P. V. (2023). Monitoring translation in all reading frames downstream of weak stop codons provides mechanistic insights into the impact of nucleotide and cellular contexts. Nucleic Acids Research, 51(1), 304–314. https://doi.org/10.1093/nar/gkac1180
9. Namy, O., Hatin, I., & Rousset, J. P. (2001). Impact of the six nucleotides downstream of the stop codon on translation termination. EMBO Reports, 2(9), 787-793. https://doi.org/10.1093/embo-reports/kve176
10. Sharma, J., Du, M., Wong, E., Mutyam, V., Li, Y., Chen, J., Wangen, J., Thrasher, K., Fu, L., Peng, N., Tang, L., Liu, K., Mathew, B., Bostwick, R. J., Augelli-Szafran, C. E., Bihler, H., Liang, F., Mahiou, J., Saltz, J., Rab, A., Hong, J., Sorscher, E. J., Mendenhall, E. M., Coppola, C. J., Keeling, K. M., Green, R., Mense, M., Suto, M. J., Rowe, S. M., & Bedwell, D. M. (2021). A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion. Nature Communications, 12(1), 4358. https://doi.org/10.1038/s41467-021-24575-x
11. Skuzeski, J. M., Nichols, L. M., Gesteland, R. F., & Atkins, J. F. (1991). The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons. Journal of Molecular Biology, 218(2), 365-373. https://doi.org/10.1016/0022-2836(91)90718-L