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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). 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.

Abstract Bifunctional stop codons that have both translation and termination functions in the same species are important for understanding the evolution and function of genetic codes in living organisms. Considering the high frequency of bifunctional codons but limited number of available genomes in ciliates, we de novo sequenced seven representative ciliate genomes to explore the evolutionary history of stop codons. We further propose a stop codon reassignment quantification method (stopCR) that can identify bifunctional codons and measure their frequencies in various eukaryotic organisms. Using our newly developed method, we found two previously undescribed genetic codes, illustrating the prevalence of bifunctional stop codons in ciliates. Overall, evolutionary genomic analyses suggest that gain or loss of reassigned stop codons in ciliates is shaped by their living environment, the eukaryotic release factor 1, and suppressor tRNAs. This study provides novel clues about the functional diversity and evolutionary history of stop codons in eukaryotic organisms.

Biomolecular condensation via liquid–liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is proposed to control critical cellular functions, whereas aberrant phase transitions are associated with a range of neurodegenerative diseases. Here, we show that a disease-associated stop codon mutation of the prion protein (PrP) at tyrosine 145 (Y145Stop), resulting in a truncated, highly disordered, N-terminal IDR, spontaneously phase-separates into dynamic liquid-like droplets. Phase separation of this highly positively charged N-terminal segment is promoted by the electrostatic screening and a multitude of weak, transient, multivalent, intermolecular interactions. Single-droplet Raman measurements, in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies, revealed a highly mobile internal organization within the liquid-like condensates. The phase behavior of Y145Stop is modulated by RNA. Lower RNA:protein ratios promote condensation at a low micromolar protein concentration under physiological conditions. At higher concentrations of RNA, phase separation is abolished. Upon aging, these highly dynamic liquid-like droplets gradually transform into ordered, β-rich, amyloid-like aggregates. These aggregates formed via phase transitions display an autocatalytic self-templating characteristic involving the recruitment and binding-induced conformational conversion of monomeric Y145Stop into amyloid fibrils. In contrast to this intrinsically disordered truncated variant, the wild-type full-length PrP exhibits a much lower propensity for both condensation and maturation into amyloids, hinting at a possible protective role of the C-terminal domain. Such an interplay of molecular factors in modulating the protein phase behavior might have much broader implications in cell physiology and disease.

Summary Cytosine base editors (CBEs) can install a predefined stop codon at the target site, representing a more predictable and neater method for creating genetic knockouts without altering the genome size. Due to the enhanced predictability of the editing outcomes, it is also more efficient to obtain homozygous mutants in the first generation. With the recent advancement of CBEs on improved editing activity, purify and specificity in plants and animals, base editing has become a more appealing technology for generating knockouts. However, there is a lack of design tools that can aid the adoption of CBEs for achieving such a purpose, especially in plants. Here, we developed a user‐friendly design tool named CRISPR‐BETS (base editing to stop), which helps with guide RNA (gRNA) design for introducing stop codons in the protein‐coding genes of interest. We demonstrated in rice and tomato that CRISPR‐BETS is easy‐to‐use, and its generated gRNAs are highly specific and efficient for generating stop codons and obtaining homozygous knockout lines. While we tailored the tool for the plant research community, CRISPR‐BETS can also serve non‐plant species.

Translational stop codon readthrough emerged as a major regulatory mechanism affecting hundreds of genes in animal genomes, based on recent comparative genomics and ribosomal profiling evidence, but its evolutionary properties remain unknown. Here, we leverage comparative genomic evidence across 21 Anopheles mosquitoes to systematically annotate readthrough genes in the malaria vector Anopheles gambiae, and to provide the first study of abundant readthrough evolution, by comparison with 20 Drosophila species. Using improved comparative genomics methods for detecting readthrough, we identify evolutionary signatures of conserved, functional readthrough of 353 stop codons in the malaria vector, Anopheles gambiae, and of 51 additional Drosophila melanogaster stop codons, including several cases of double and triple readthrough and of readthrough of two adjacent stop codons. We find that most differences between the readthrough repertoires of the two species arose from readthrough gain or loss in existing genes, rather than birth of new genes or gene death; that readthrough-associated RNA structures are sometimes gained or lost while readthrough persists; that readthrough is more likely to be lost at TAA and TAG stop codons; and that readthrough is under continued purifying evolutionary selection in mosquito, based on population genetic evidence. We also determine readthrough-associated gene properties that predate readthrough, and identify differences in the characteristic properties of readthrough genes between clades. We estimate more than 600 functional readthrough stop codons in mosquito and 900 in fruit fly, provide evidence of readthrough control of peroxisomal targeting, and refine the phylogenetic extent of abundant readthrough as following divergence from centipede.

Abstract In bacteria stop codons are recognized by one of two class I release factors (RF1) recognizing TAG, RF2 recognizing TGA, and TAA being recognized by both. Variation across bacteria in the relative abundance of RF1 and RF2 is thus hypothesized to select for different TGA/TAG usage. This has been supported by correlations between TAG:TGA ratios and RF1:RF2 ratios across multiple bacterial species, potentially also explaining why TAG usage is approximately constant despite extensive variation in GC content. It is, however, possible that stop codon trends are determined by other forces and that RF ratios adapt to stop codon usage, rather than vice versa. Here, we determine which direction of the causal arrow is the more parsimonious. Our results support the notion that RF1/RF2 ratios become adapted to stop codon usage as the same trends, notably the anomalous TAG behavior, are seen in contexts where RF1:RF2 ratios cannot be, or are unlikely to be, causative, that is, at 3′untranslated sites never used for translation termination, in intragenomic analyses, and across archaeal species (that possess only one RF1). We conclude that specifics of RF biology are unlikely to fully explain TGA/TAG relative usage. We discuss why the causal relationships for the evolution of synonymous stop codon usage might be different from those affecting synonymous sense codon usage, noting that transitions between TGA and TAG require two-point mutations one of which is likely to be deleterious.

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein–protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

De novo protein-coding innovations sometimes emerge from ancestrally noncoding DNA, despite the expectation that translating random sequences is overwhelmingly likely to be deleterious. The “preadapting selection” hypothesis claims that emergence is facilitated by prior, low-level translation of noncoding sequences via molecular errors. It predicts that selection on polypeptides translated only in error is strong enough to matter and is strongest when erroneous expression is high. To test this hypothesis, we examined noncoding sequences located downstream of stop codons (i.e., those potentially translated by readthrough errors) in Saccharomyces cerevisiae genes. We identified a class of “fragile” proteins under strong selection to reduce readthrough, which are unlikely substrates for co-option. Among the remainder, sequences showing evidence of readthrough translation, as assessed by ribosome profiling, encoded C-terminal extensions with higher intrinsic structural disorder, supporting the preadapting selection hypothesis. The cryptic sequences beyond the stop codon, rather than spillover effects from the regular C-termini, are primarily responsible for the higher disorder. Results are robust to controlling for the fact that stronger selection also reduces the length of C-terminal extensions. These findings indicate that selection acts on 3′ UTRs in Saccharomyces cerevisiae to purge potentially deleterious variants of cryptic polypeptides, acting more strongly in genes that experience more readthrough errors.

We report the crystal structure of a translation termination complex formed by the Thermus thermophilus 70S ribosome bound with release factor RF2, in response to a UAA stop codon, solved at 3 Å resolution. The backbone of helix α5 and the side chain of serine of the conserved SPF motif of RF2 recognize U1 and A2 of the stop codon, respectively. A3 is unstacked from the first 2 bases, contacting Thr-216 and Val-203 of RF2 and stacking on G530 of 16S rRNA. The structure of the RF2 complex supports our previous proposal that conformational changes in the ribosome in response to recognition of the stop codon stabilize rearrangement of the switch loop of the release factor, resulting in docking of the universally conserved GGQ motif in the PTC of the 50S subunit. As seen for the RF1 complex, the main-chain amide nitrogen of glutamine in the GGQ motif is positioned to contribute directly to catalysis of peptidyl-tRNA hydrolysis, consistent with mutational studies, which show that most side-chain substitutions of the conserved glutamine have little effect. We show that when the H-bonding capability of the main-chain N-H of the conserved glutamine is eliminated by substitution with proline, peptidyl-tRNA esterase activity is abolished, consistent with its proposed role in catalysis.

Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes. Amino acids are the building blocks of proteins, and the order of the amino acids in a protein is determined by the order in which ‘codons’ appear in a messenger RNA molecule. Most codons represent a specific amino acid, but there are also three stop codons that are used to mark the end of a protein. When the cellular machinery that ‘translates’ the messenger RNA molecule into a protein encounters a stop codon, it stops and releases the completed protein. Sometimes, however, the stop codon is not interpreted as a stop signal, and the translation of the messenger RNA molecule continues until another stop codon is encountered. This process is known as readthrough. Some organisms, in particular viruses and fungi, use readthrough to produce a wider range of proteins than their genomes would otherwise allow. While readthrough also occurs in higher organisms such as mammals, it is not known if the resulting proteins perform extra functions that the original protein does not perform. A number of factors affect whether readthrough occurs when an mRNA template is being translated. For example, each of the three stop codons has a different likelihood of having its stop signal misinterpreted, and the mRNA sequence that surrounds the stop codon can also affect the likelihood of readthrough. Schueren et al. have developed a computational model that estimates how common this form of translational readthrough is in the human genome. The model was based on the identity of the stop codons themselves and the surrounding mRNA sequence. This model was then combined with another model that identifies proteins that are targeted to a structure inside a cell called the peroxisome, which is where a number of essential energy-releasing reactions take place. The combined model enabled Schueren et al. to identify proteins that both perform functions in the peroxisome and are likely to be formed by readthrough. The combined model suggested a protein that is a part of lactate dehydrogenase: an enzyme that speeds up chemical reactions that are important for the cell to produce energy. Low levels of lactate dehydrogenase had previously been found in the peroxisome, despite it apparently lacking a specific sequence of amino acids that proteins need to have to enter the peroxisome. However, Schueren et al. confirmed experimentally that readthrough does occur for the lactate dehydrogenase component identified by the model, revealing that it contains a ‘hidden’ peroxisome-targeting region. Furthermore, when more translational readthrough occurred, more lactate dehydrogenase was found in the peroxisomes. This unusual way that lactate dehydrogenase enters the peroxisome is an example of how the cell optimizes the used of the genetic information encoded in the genome and in messenger RNA. Translational readthrough always ensures that a certain proportion of lactate dehydrogenase will be brought to the peroxisome. The computational model developed here will be a valuable tool to identify other such proteins produced from genomes, including the human genome and those of other species.