Insights on Nonsense Suppression Therapy with Translatomics
Nonsense mutations, single nucleotide changes which replaces a codon that encodes for an amino acid with a stop codon resulting in truncated protein products, give rise to approximately 11% of inherited diseases in humans. Nonsense suppression therapeutics may act as viable treatments for such diseases by allowing ribosomes to read through premature termination codons and synthesize the full functional form of affected proteins. Ribosome profiling studies can allow for in-depth analysis of ribosome occupancy at normal and premature termination codons, features of mRNA sequences that influence readthrough, and hence termination efficiencies, and stop codon readthrough rates. Here, we’ve selected some research articles that discuss nonsense suppression therapy and feature ribosome profiling, highlighting the unique insights gained.
Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster
eLife, 2013, 2, p.e01179
Dunn, J.G., Foo, C.K., Belletier, N.G., Gavis, E.R. and Weissman, J.S.
Stop codon readthrough occurs when ribosomes fail to terminate at stop codons, allowing for elongation of the nascent peptide. This phenomenon has proved critical to the proteomic diversity of many animal and plant viruses and is also thought to play a functionally important role for a subset of genes in eukaryotic organisms. The C-terminal extension of a protein following readthrough of its annotated stop codon can lead to changes in its activity, stability and/or localization. In addition, readthrough of premature stop codons (PTCs) can suppress pathological phenotypes arising from translation of truncated variants and ameliorate the need for nonsense-mediated decay of associated transcripts.
While analysing phylogenetic signatures of amino acid conservation in the region between the annotated and next in-frame stop codons allows for prediction of phylogenetically conserved readthrough events, such an approach has limited capacity when it comes to more evolutionary recent events. Ribosome profiling is uniquely suited to explore such events, by identifying the tissues or cell types in which readthrough occurs, calculating the extent of readthrough at a given stop codon, and investigating whether these processes are differentially regulated. Here, the authors utilize a modified ribosome profiling protocol to provide the first genome-wide experimental analysis of readthrough in D. melanogaster.
- By examining the locations of ribosomes along mRNAs, the authors identify more than 300 readthrough events not predicted by phylogenetic approaches. They show that these novel extensions are of recent evolutionary origin.
- Specific examples of both novel and conserved extensions are validated to show they can produce stable protein products in a regulated manner, and contain functional subcellular localization signals.
- It was also demonstrated that readthrough occurs at many loci in yeast and in primary human foreskin fibroblasts. The authors suggest that readthrough is both a ubiquitous feature of eukaryotic translation and a novel mechanism in regulating gene expression.
This is the first paper to showcase the ability of Ribosome Profiling to effectively identify readthrough events genome wide in a eukaryotic organism. The resulting findings demonstrate that such events provide an important evolutionary means for genes to acquire new functions.
Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides
eLife, 2020, 9, p.e52611
Wangen, J.R. and Green, R.
Stop codon readthrough occurs when ribosomes miscode at a stop codon and continue translating. While translation termination is typically the dominant reaction at stop codons, termination efficiencies can vary depending on the codon context. In addition to the identity of the stop codon, the surrounding sequence context, proximal RNA structures, RNA modifications and near-cognate aminoacyl-tRNA availability also influences the probability of whether ribosomes terminate translation at a transcript’s annotated stop or continue translating, either because of readthrough or frameshifting (where the ribosome slides on an mRNA, changing the frame of translation).
Readthrough events have shown to be of therapeutic interest when a premature termination codon (PTC) is found in an essential gene. Nonsense mutations can hinder translation by inserting a PTC within the coding sequence of a gene. Ribosomes terminate translation at PTCs resulting in a truncated peptide, which usually comes with loss of function and potential dominant-negative effects. Compounds which act as nonsense suppression agents have the potential to increase rates of readthrough at PTCs on target genes, restoring translation of full-length functional proteins. Here, the authors investigated a class of nonsense suppression agents known as aminoglycosides (AG). AGs are well-characterized in bacterial systems, where they bind the decoding center of the ribosome and promote miscoding. Ribosome profiling, an approach which provides an unbiased examination of translation termination for all stop codons in their native sequence contexts, was carried out to examine the activities of AGs in promoting readthrough of stop codons genome-wide.
- Broad stimulation of stop codon readthrough was observed following treatment with AGs, especially with aminoglycoside G418. A general role for termination codon identity, as well as surrounding sequence contexts in determining genome-wide rates of stop codon readthrough were observed.
- Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context were all found to influence the probability of stop codon readthrough.
- G418 encourages readthrough at multiple classes of stop codons including PTCs, normal termination codons (NTCs), and 3′UTR termination codons (3’TCs). G418 was found to more potently induce readthrough of 3′TCs relative to NTCs, which was attributed to an enrichment of A’s and U’s in the stop codon context of 3’TCs.
- Several biological processes were found to be disrupted by high levels of G418-induced stop codon readthrough, including translation of histone mRNAs, selenoproteins, and S-adenosylmethionine decarboxylase 1 (AMD1).
The authors showed that G418 successfully induced readthrough of a PTC in the frequently mutated oncogene TP53 and partially restored levels of its full length. However, the drug also induces widespread disruptive effects on translation globally. Despite this, the Ribo-Seq data presented highlights the role of the stop codon and its surrounding context in regulating translation termination, information which will no doubt be crucial to the development of nonsense suppression therapeutics capable of selectively inducing readthrough at PTCs without disrupting translation termination globally.
Engineered transfer RNAs for suppression of premature termination codons
Nature Communications, 2019, 10(1), pp.1-11
Lueck, J.D., Yoon, J.S., Perales-Puchalt, A., Mackey, A.L., Infield, D.T., Behlke, M.A., Pope, M.R., Weiner, D.B., Skach, W.R., McCray, P.B. and Ahern, C.A.
Premature termination codons (PTCs) occur as a result of single nucleotide mutations, whereby a canonical triplet nucleotide codon is converted into one of three stop codons, e.g., TAG, TGA, or TAA. PTCs can be more deleterious than missense mutations as they lead to a loss of protein expression, as well as decreases in mRNA abundance through nonsense-mediated decay (NMD). Additionally, some truncated proteins may have a dominant negative function. PTCs are responsible for 10–15% of all inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy, and spinal muscular atrophy. Nonsense mutations are also found within the tumour suppressor genes p53 and ATM. PTC suppression during translation could be utilized to treat a variety of genetic disorders, however small molecules that promote PTC read-through have garnered mixed results in clinical trials.
Small molecules, such as aminoglycosides, dipeptides, and oxadiazoles can promote read-through of nonsense mutations. However, this approach results in the encoding of a near-cognate amino acid, effectively generating a missense mutation at the PTC, which may have deleterious effects on protein folding, trafficking, and function. The authors aimed to identify a PTC repair approach that displays the versatility of small molecule therapeutics and the precision of gene editing. They investigated tRNAs to fulfil these criteria, whereby their anticodons have been engineered via mutagenesis to recognize and suppress UGA, UAA, or UAG PTC codons.
- Here, the authors show that an anti-codon editing approach is generalizable to multiple tRNA gene families, indicating that many annotated tRNA are biologically viable.
- Many of the investigated anti-codon engineered-tRNAs (ACE-tRNAs) manifested 100 to 1000-fold suppression of PTC termination, which is significantly higher than readthrough levels observed with the aminoglycoside’s gentamycin and G418.
- These ACE-tRNAs exhibited high fidelity translation of full-length proteins, encoding for the cognate amino acid greater than 98% of the time.
- Ribosome profiling data showed that efficiency of native stop codon readthrough by most ACE-tRNAs was markedly less than the level of PTC readthrough, however some induced approximately a 2-fold increase in 3′UTR ribosome density for the cognate stop codon complimentary to the ACE-tRNA anticodon.
The authors introduce a new class of PTC therapeutic capable of producing potent and stable in-vivo PTC suppression of several disease-causing nonsense mutations that rescued the corresponding full-length proteins. Despite this, the potential for off-target readthrough exhibited by a small number of these ACE-tRNAs shows that Ribo-Seq is an essential tool for evaluating their safety for use as a therapeutic agent.
Nonsense mRNA suppression via nonstop decay
ELife, 2018, 7, p.e33292–3087
Arribere, J.A. and Fire, A.Z.
Nonsense-mediated decay (NMD) is a translational surveillance mechanism that identifies and targets deleterious products of premature stop codons. In NMD, recognition of an early stop codon destabilizes an mRNA. The NMD pathway can also contribute to pathological suppression of several disease-causing mutations, about 11% of point mutations are linked to human disease. However, understanding the downstream events leading to suppression of gene expression remains limited. Sources for the uncertainty include technical complications, such as the transient nature of RNA decay intermediates and variations in NMD machinery between organisms.
Nonstop decay (NSD) is a second translational surveillance pathway in which cells repress the activity of mRNAs lacking stop codons through both mRNA and protein decay mechanisms. These confer a functional redundancy to NSD that has hampered genetic approaches. Two factors known to play a crucial role in the NSD pathway are SKI, a multi-protein complex that facilitates 3’end degradation of mRNAs by exonucleases, and dom34/pelota, a release factor that targets stalled ribosomes. Using an adapted Ribo-Seq protocol that captures “nonstop fragments” (shorter 15-18nt fragments that represent a stalled ribosome at the 3’edge of a mRNA) on both individual and dual knockout models of these two NSD factors in Caenorhabditis elegans, the authors discovered that they play an unexpected role in the degradation of NMD generated mRNA fragments.
- The authors confirmed that NSD occurs in C. elegans and that both the SKI complex and PELO-1 (the functional ortholog of dom34/pelota in C. elegans) are important to its function.
- The distribution of ribosome protected “nonstop” fragments at the 3’end of transcripts on lacking a stop codon in PELO-1 and PELO-1/SKI knockouts supports a model for NSD conserved between S. cerevisiae and C. elegans where PELO-1 rescues ribosomes stalled on 3’-truncated RNA fragments, and SKI ensures efficient nonstop mRNA clearance.
- An accumulation of “nonstop” fragments was also observed on transcripts with truncated stop codons indicating a role for PELO-1 in releasing stalled ribosomes resistant to normal eukaryotic Release Factor-1-mediated translation termination.
- The authors also observed an accumulation of these “nonstop” fragments at and upstream of premature stop codons, supporting a model wherein NMD funnels into the NSD pathway in C. elegans.
This paper provides strong evidence for the existence of a link between NMD and NSD pathways, advancing understanding of these molecular processes which in turn opens up the potential for therapeutic targeting of these pathways.
Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae
PLOS Genetics, 2021, 17(4): e1009538–3087
Mangkalaphiban, K., He, F., Ganesan, R., Wu, C., Baker, R. and Jacobson, A.
The release factor eRF1 terminates mRNA translation by interacting with a stop codon in the ribosomal A site. In some cases, a near-cognate tRNA can base pair with a stop codon, allowing the elongation phase of protein synthesis to proceed, a phenomenon called stop codon readthrough. Such readthrough can be influenced by factors other than the identity of the stop codon, as seen in studies with reporter systems. Examples of these include the two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon, as well as stem-loop structures in the mRNA 3’-UTR. It remains to be seen if these elements are critical at a genome-wide level. Other factors near the stop codon may also have a significant effect on translation termination and readthrough efficiencies in vivo.
Ribosome profiling in yeast, human, and fly cells have already shown that readthrough is more common than previously thought, and that efficiencies for distinct mRNAs can vary by more than 100-fold. Translation termination efficiency can be susceptible to transcript-specific regulation, and certain cis-acting mRNA sequences may prove important in gaining further knowledge on this regulation. Readthrough of individual mRNAs has been uncovered in previous ribosome profiling studies, however further examination is needed to explore the associations between readthrough efficiency and cis-acting mRNA sequences. Here, the authors carried out ribosome profiling of yeast cells expressing wild-type or temperature-sensitive eRF1. Bioinformatics approaches were designed to calculate readthrough efficiency, and to identify mRNA and peptide features that influence efficiency.
- The authors found that the stop codon (nt +1 to +3), the nucleotide following it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects.
- The presence of four codons (CGU, GCU, UUA, and UUC) in the p-site were found to be associated with lower readthrough efficiencies, while five (AUA, ACA, ACC, CUG, and GAC) associated with higher readthrough efficiencies. Despite this, the identity of the last amino acid residue was not found to influence readthrough. Instead, the authors suggest a synergistic effect between specific combinations of nucleotide and tRNA properties is likely influencing readthrough.
- Additionally, it was found that low readthrough genes have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while the opposite was observed in wild-type cells. The authors postulated that the absence of eRF1 activity highlights the role of poly(A)-binding protein (PABP) in recruiting other release factors to facilitate termination, with the efficiency of this action being dependent on proximity to the stop codon. The effect of this feature was masked in WT cells where eRF1 was fully functional.
While confirming the roles of known regulatory elements in genome-wide regulation, the paper also identified several new mRNA features that appear to play a role in regulating translation termination and readthrough. Deeper understanding of these regulatory elements could be useful in designing therapeutic approaches for the large number of diseases caused by nonsense mutations.
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