Suppression of premature termination codons as a therapeutic approach

In this review, we describe our current understanding of translation termination and pharmacological agents that influence the accuracy of this process. A number of drugs have been identified that induce suppression of translation termination at in-frame premature termination codons (PTCs; also known as nonsense mutations) in mammalian cells. We discuss efforts to utilize these drugs to suppress disease-causing PTCs that result in the loss of protein expression and function. In-frame PTCs represent a genotypic subset of mutations that make up ~11% of all known mutations that cause genetic diseases, and millions of patients have diseases attributable to PTCs. Current approaches aimed at reducing the efficiency of translation termination at PTCs (referred to as PTC suppression therapy) have the goal of alleviating the phenotypic consequences of a wide range of genetic diseases. Suppression therapy is currently in clinical trials for treatment of several genetic diseases caused by PTCs, and preliminary results suggest that some patients have shown clinical improvements. While current progress is promising, we discuss various approaches that may further enhance the efficiency of this novel therapeutic approach.     

Suppression of premature termination codons as a therapeutic approach

In this review, we describe our current understanding of translation termination and pharmacological agents that influence the accuracy of this process. A number of drugs have been identified that induce suppression of translation termination at in-frame premature termination codons (PTCs; also known as nonsense mutations) in mammalian cells. We discuss efforts to utilize these drugs to suppress disease-causing PTCs that result in the loss of protein expression and function. In-frame PTCs represent a genotypic subset of mutations that make up ~11% of all known mutations that cause genetic diseases, and millions of patients have diseases attributable to PTCs. Current approaches aimed at reducing the efficiency of translation termination at PTCs (referred to as PTC suppression therapy) have the goal of alleviating the phenotypic consequences of a wide range of genetic diseases. Suppression therapy is currently in clinical trials for treatment of several genetic diseases caused by PTCs, and preliminary results suggest that some patients have shown clinical improvements. While current progress is promising, we discuss various approaches that may further enhance the efficiency of this novel therapeutic approach.     

Rescue of non-sense mutated p53 tumor suppressor gene by aminoglycosides

Mutation-based treatments are a new development in genetic medicine, in which the nature of the mutation dictates the therapeutic strategy. Interest has recently focused on diseases caused by premature termination codons (PTCs). Drugs inducing the readthrough of these PTCs restore the production of a full-length protein. In this study, we explored the possibility of using aminoglycoside antibiotics to induce the production of a full-length functional p53 protein from a gene carrying a PTC. We identified a human cancer cell line containing a PTC, for which high levels of readthrough were obtained in the presence of aminoglycosides. Using these cells, we demonstrated that aminoglycoside treatment stabilized the mutant mRNA, which would otherwise have been degraded by non-sense-mediated decay, resulting in the production of a functional full-length p53 protein. Finally, we showed that aminoglycoside treatment decreased the viability of cancer cells specifically in the presence of nonsense-mutated p53 gene. These results open possibilities of developing promising treatments of cancers linked with non-sense mutations in tumor suppressor genes. They show that molecules designed to induce stop-codon readthrough can be used to inhibit tumor growth and offer a rational basis for developing new personalized strategies that could diversify the existing arsenal of cancer therapies.     

Readthrough compounds for nonsense mutations: bridging the translational gap

Approximately 10% of all pathological mutations are nonsense mutations that are responsible for several severe genetic diseases for which no treatment regimens are currently available. The most widespread strategy for treating nonsense mutations is by enhancing ribosomal readthrough of premature termination codons (PTCs) to restore the production of the full-length protein. In the past decade several compounds with readthrough potential have been identified. However, although preclinical results on these compounds are promising, clinical studies have not yielded positive outcomes. We review preclinical and clinical research related to readthrough compounds and characterize factors that contribute to the observed translational gap.     

tRNA抑制子对转录因子NKX2.5提前终止密码子的通读

人类NKX2.5基因(NK2 homeobox 5,NKX2.5)提前终止密码子(Premature termination codon,PTC)突变会引起房间隔缺损、房室传导阻滞等先天性心脏病。目前,已报道的NKX2.5 PTC突变有8个(E109X、Q149X、Q170X、Q187X、Q198X、Y256X、Y259X和C264X)。为了检测tRNA抑制子是否对PTC突变诱导通读产生有功能的全长蛋白,文章将8个NKX2.5 PTC突变克隆到pcDNA3.1(-)载体,将NKX2.5全长和E109X、Q149X及C264X克隆到pEGFP-N1载体,形成NKX2.5-EGFP融合质粒。将NKX2.5-EGFP与对应的tRNA抑制子质粒分别或共转染后观察绿色荧光数量定性判断tRNA抑制子是否诱导通读。Western blotting检测通读后全长蛋白和截短蛋白表达并计算通读效率。Real-time PCR检测NKX2.5下游重要调控基因Cx43 mRNA的表达判断通读后蛋白功能。结果表明,文章成功构建了8个基于pcDNA3.1(-)的NKX2.5表达质粒、4个基于pEGFP-N1的质粒;tRNA抑制子tRNA am能有效通读Q149X、Q170X、Q187X和Q198X,且对后三者的通读效率均在50%以上;tRNA op能有效通读C264X,通读效率约50%左右;tRNA oc不能通读NKX2.5 PTC突变;各通读后样本Cx43 mRNA相对表达量增加7%~41.7%;tRNA am和tRNA op能有效通读NKX2.5 PTC突变,产生具有功能的全长蛋白,但tRNA抑制子对细胞的其他影响还不明确,有待于进一步观察。     

Applying nonsense-mediated mRNA decay research to the clinic: progress and challenges

Premature termination codons (PTCs) are equivalent to nonsense sequences. They encode no amino acid, and their presence precludes the synthesis of full-length proteins. Furthermore, the resulting truncated proteins, if synthesized and stable, are likely to be non-functional or might even be deleterious to cellular metabolism. Approximately one third of genetic and acquired diseases are due to PTCs. In fact, PTCs are apt to cause at least some cases of all diseases that involve protein insufficiency. Cells have evolved a way to eliminate mRNAs that contain PTCs using a mechanism called nonsense-mediated mRNA decay (NMD). Here, we will review how to determine which PTCs elicit NMD, what is currently known about the mechanism of NMD, and additional information that is pertinent to establishing therapies for PTC-associated diseases.     

RNA splicing promotes translation and RNA surveillance

Aberrant mRNAs harboring premature termination codons (PTCs or nonsense codons) are degraded by the nonsense-mediated mRNA decay (NMD) pathway. mRNAs transcribed from genes that naturally acquire PTCs during lymphocyte development are strongly downregulated by PTCs. Here we show that a signal essential for this robust mRNA downregulatory response is efficient RNA splicing. Strong mRNA downregulation can be conferred on a poor NMD substrate by either strengthening its splicing signals or removing its weak introns. Efficient splicing also strongly promotes translation, providing a molecular explanation for enhanced NMD and suggesting that efficient splicing may have evolved to enhance both protein production and RNA surveillance. Our results suggest simple approaches for increasing protein expression from expression vectors and treating human genetic diseases caused by nonsense and frameshift mutations.     

Statistical analysis of readthrough levels for nonsense mutations in mammalian cells reveals a major determinant of response to gentamicin

The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the -1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable.     

Rescue of nonsense mutations by amlexanox in human cells

ABSTRACT: BACKGROUND: Nonsense mutations are at the origin of many cancers and inherited genetic diseases. The consequence of nonsense mutations is often the absence of mutant gene expression due to the activation of an mRNA surveillance mechanism called nonsense-mediated mRNA decay (NMD). Strategies to rescue the expression of nonsense-containing mRNAs have been developed such as NMD inhibition or nonsense mutation readthrough. METHODS: Using a dedicated screening system, we sought molecules capable to block NMD. Additionally, 3 cell lines derived from patient cells and harboring a nonsense mutation were used to study the effect of the selected molecule on the level of nonsense-containing mRNAs and the synthesis of proteins from these mutant mRNAs. RESULTS: We demonstrate here that amlexanox, a drug used for decades, not only induces an increase in nonsense-containing mRNAs amount in treated cells, but also leads to the synthesis of the full-length protein in an efficient manner. We also demonstrated that these full length proteins are functional. CONCLUSIONS: As a result of this dual activity, amlexanox may be useful as a therapeutic approach for diseases caused by nonsense mutations.     

Allele-specific therapy: suppression of nonsense mutations by readthrough inducers

Ten percent of human hereditary diseases are linked to nonsense mutations (premature termination codon). These mutations lead to premature translation termination, trigger the synthesis of a truncated protein and possibly lead to mRNA degradation by the NMD pathway (nonsense mediated mRNA decay). For the past ten years, therapeutic strategies have emerged which attempt to use molecules that facilitate tRNA incorporation at premature stop codon (readthrough), thus allowing for the synthesis of a full length protein. Molecules currently used for this approach are mostly aminoglycoside antibiotics (gentamicin, amikacin…) that bind the decoding center of the ribosome. This therapeutic approach has been studied for various genetic diseases including Duchenne muscular dystrophy (DMD) and cystic fibrosis. The feasibility of this approach depends on induced readthrough level, mRNA quantity, re-expressed protein functionality and characteristics of each disease.     

Chemical-Induced Read-Through at Premature Termination Codons Determined by a Rapid Dual-Fluorescence System Based on S. cerevisiae

Nonsense mutations generate in-frame stop codons in mRNA leading to a premature arrest of translation. Functional consequences of premature termination codons (PTCs) include the synthesis of truncated proteins with loss of protein function causing severe inherited or acquired diseases. A therapeutic approach has been recently developed that is based on the use of chemical agents with the ability to suppress PTCs (read-through) restoring the synthesis of a functional full-length protein. Research interest for compounds able to induce read-through requires an efficient high throughput large scale screening system. We present a rapid, sensitive and quantitative method based on a dual-fluorescence reporter expressed in the yeast Saccharomyces cerevisiae to monitor and quantitate read-through at PTCs. We have shown that our novel system works equally well in detecting read-through at all three PTCs UGA, UAG and UAA.     

Deciphering the molecular mechanism of stop codon readthrough

Recognition of the stop codon by the translation machinery is essential to terminating translation at the right position and to synthesizing a protein of the correct size. Under certain conditions, the stop codon can be recognized as a coding codon promoting translation, which then terminates at a later stop codon. This event, called stop codon readthrough, occurs either by error, due to a dedicated regulatory environment leading to generation of different protein isoforms, or through the action of a readthrough compound. This review focuses on the mechanisms of stop codon readthrough, the nucleotide and protein environments that facilitate or inhibit it, and the therapeutic interest of stop codon readthrough in the treatment of genetic diseases caused by nonsense mutations.     

The paradox of viable <Emphasis Type="Italic">sup45</Emphasis> STOP mutations: a necessary equilibrium between translational readthrough,activity and stability of the protein

The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q → TAA, 242R → TGA, 317L → TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells.     

Yeast as a model for studying human neurodegenerative disorders

Protein misfolding and aggregation are central events in many disorders including several neurodegenerative diseases. This suggests that alterations in normal protein homeostasis may contribute to pathogenesis, but the exact molecular mechanisms involved are still poorly understood. The budding yeast Saccharomyces cerevisiae is one of the model systems of choice for studies in molecular medicine. Modeling human neurodegenerative diseases in this simple organism has already shown the incredible power of yeast to unravel the complex mechanisms and pathways underlying these pathologies. Indeed, this work has led to the identification of several potential therapeutic targets and drugs for many diseases, including the neurodegenerative diseases. Several features associated with these diseases, such as formation of protein aggregates, cellular toxicity mediated by misfolded proteins, oxidative stress and hallmarks of apoptosis have been faithfully recapitulated in yeast, enabling researchers to take advantage of this powerful model to rapidly perform genetic and compound screens with the aim of identifying novel candidate therapeutic targets and drugs. Here we review the work undertaken to model human brain disorders in yeast, and how these models provide insight into novel therapeutic approaches for these diseases.     

Functional Translational Readthrough: A Systems Biology Perspective

Translational readthrough (TR) has come into renewed focus because systems biology approaches have identified the first human genes undergoing functional translational readthrough (FTR). FTR creates functional extensions to proteins by continuing translation of the mRNA downstream of the stop codon. Here we review recent developments in TR research with a focus on the identification of FTR in humans and the systems biology methods that have spurred these discoveries.     

无义介导的mRNA降解机制及其在单基因遗传病中的作用

无义介导的mRNA降解(Nonsense-mediated mRNA decay,NMD)是一种广泛存在于真核生物细胞中的mRNA质量监控机制。该机制通过识别和降解含有提前终止密码子(Premature translational-termination codon,PTC)的转录产物防止有潜在毒性的截短蛋白的产生。据估计,约1/3的遗传性疾病是由提前终止密码子引起的,而NMD作用通常会改变某些遗传病的临床症状或遗传方式。文章主要综述了人体细胞中NMD对底物的识别及其作用机制,并以几种单基因遗传病为例探讨其对这些疾病表型的影响,表明NMD作用机制的进一步揭示将有助于单基因遗传病发病机制的阐明及治疗方法的改进。     

Generation of stable cell lines using readthrough expression from lentiviral integration

Lentiviral infection is often used to integrate genetic material into cells to stably express transgenes of interest. Depending on the location of integration into the host genome, readthrough expression of the lentiviral cargo can occur via an upstream endogenous promoter, which is typically an unwanted phenomenon because it can result in dysfunctional expression. The purpose of this study was to demonstrate that readthrough expression can be a wanted phenomenon for expressing functional proteins while at the same time reducing the size of the lentiviral transfer plasmid. Readthrough expression was used to generate HEK293 cell lines stably expressing fluorescent reporter proteins, reporter protein-antibiotic resistance fusion proteins for selection, and the vascular endothelial growth factor receptor 2. The generated proteins were all functional, as demonstrated by their ability to fluoresce, confer antibiotic resistance, and participate in receptor-mediated signalling, respectively. Therefore, we suggest that the mechanism of readthrough expression may have further applications in the expression of larger genes or genetic circuits (e.g. cell-based therapeutics), where the lentiviral cargo limit is stretched to the maximum.

     

Listening to silence and understanding nonsense: exonic mutations that affect splicing

Point mutations in the coding regions of genes are commonly assumed to exert their effects by altering single amino acids in the encoded proteins. However, there is increasing evidence that many human disease genes harbour exonic mutations that affect pre-mRNA splicing. Nonsense, missense and even translationally silent mutations can inactivate genes by inducing the splicing machinery to skip the mutant exons. Similarly, coding-region single-nucleotide polymorphisms might cause phenotypic variability by influencing splicing accuracy or efficiency. As the splicing mechanisms that depend on exonic signals are elucidated, new therapeutic approaches to treating certain genetic diseases can begin to be explored.     

mRNA quality control: an ancient machinery recognizes and degrades mRNAs with nonsense codons

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway which ensures the rapid degradation of mRNAs containing premature translation termination codons (PTCs or nonsense codons), thereby preventing the accumulation of truncated and potentially harmful proteins. In this way, the NMD pathway contributes to suppressing or exacerbating the clinical manifestations of specific human genetic disorders. Studies in model organisms have led to the identification of the effectors of the NMD pathway, and illuminated the mechanisms by which premature stops are discriminated from natural stops, so that only the former trigger rapid mRNA degradation. These studies are providing important insights that will aid the development of new treatments for at least some human genetic diseases.