Role of ribosome recycling factor in natural termination and translational coupling as a ribosome releasing factor.

The role of ribosome recycling factor (RRF) of E. coli was studied in vivo and in vitro. We used the translational coupling without the Shine-Dalgarno sequence of downstream ORF (d-ORF) as a model system of the RRF action in natural termination of protein synthesis. For the in vivo studies we used the translational coupling by the adjacent coat and lysis genes of RNA phage GA sharing the termination and initiation (UAAUG) and temperature sensitive RRF. The d-ORF translation was measured by the expression of the reporter lacZ gene connected to the 5'-terminal part of the lysis gene. The results showed that more ribosomes which finished upstream ORF (u-ORF) reading were used for downstream reading when RRF was inactivated. The in vitro translational coupling studies with 027mRNA having the junction sequence UAAUG with wild-type RRF were carried out with measuring amino acids incorporation. The results showed that ribosomes released by RRF read downstream from AUG of UAAUG. In the absence of RRF, ribosomes read downstream in frame with UAA. These in vivo and in vitro studies indicate that RRF releases ribosomes from mRNA at the termination codon of u-ORF. Furthermore, the non-dissociable ribosomes read downstream from AUG of UAAUG with RRF in vitro. This suggests that complete ribosomal splitting is not required for ribosome release by RRF in translational coupling. The data are consistent with the interpretation that RRF functions mostly as a ribosome releasing factor rather than ribosome splitting factor. Additionally, the in vivo studies showed that short (less than 5 codons) u-ORF inhibited d-ORF reading by ribosomes finishing u-ORF reading, suggesting that the termination process in short ORF is not similar to that in normal ORF. This means that all the preexisting studies on RRF with short mRNA may not represent what goes on in natural termination step.

Recognition of 3' nucleotide context and stop codon readthrough are determined during mRNA translation elongation.

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3' contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3' stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3' nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3' nucleotides. Moreover, the efficiency of translation termination in weak 3' contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3' nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.

[Role of Proteins Interacting with the eRF1 and eRF3 Release Factors in the Regulation of Translation and Prionization].

The review discusses the role that proteins interacting with the translation termination factors eRF1 and eRF3 play in the control of protein synthesis and prionization. These proteins interact not only with each other, but also with many other proteins involved in controlling the efficiency of translation termination, and associate translation termination with other cell processes. The termination of translation is directly related not only to translation re-initiation and ribosome recycling, but also to mRNA stability and protein quality control. This connection is ensured by the interaction of eRF1 and eRF3 with proteins participating in various cell metabolic processes, such as mRNA transport from the nucleus into the cytoplasm (Dbp5/DDX19 and Gle1), ribosome recycling (Rli1/ABCE1), mRNA degradation (Upf proteins), and translation initiation (Pab1/PABP). In addition to genetic control, there is epigenetic control of translation termination. This mechanism is associated with prion polymerization of the Sup35 protein to form the [PSI^(+)] prion. The maintenance of the [PSI^(+)] prion, like other yeast prions, requires the operation of a system of molecular chaperones and protein sorting factors. The review considers in detail the interaction of the translation termination factors with proteins involved in various cellular processes.

KEPI plays a negative role in the repression that accompanies translational inhibition guided by the uORF element of human CHOP transcript during stress response.

Translation of the downstream coding sequence of some mRNAs may be repressed by the upstream open reading frame (uORF) at their 5'-end. The mechanism underlying this uORF-mediated translational inhibition (uORF-MTI) is not fully understood in vivo. Recently, it was found that zebrafish Endouc or its human orthologue ENDOU (Endouc/ENDOU) plays a positive role in repressing the uORF-MTI of human CHOP (uORFchop-MTI) during stress by blocking its activity However, the repression of uORFchop-MTI assisted by an as-yet unidentified negative effector remains to be elucidated. Compared to the upregulated CHOP transcript, we herein report that the kepi (kinase-enhanced PP1 inhibitor) transcript was downregulated in the zebrafish embryos treated with both heat shock and hypoxia. Quantitative RT-PCR also revealed that the level of kepi mRNA was noticeably decreased in both heat-shock-treated and hypoxia-exposed embryos. When kepi mRNA was microinjected into the one-celled embryos from transgenic line huORFZ, the translation of downstream GFP reporter controlled by the uORFchop-MTI was reduced in the hypoxia-exposed embryos. In contrast, when kepi was knocked down by injection of antisense Morpholino oligonucleotide, the translation of downstream GFP reporter was induced and expressed in the brain and spinal cord of injected embryos in the absence of stress. During normal condition, overexpression of KEPI increased eIF2α phosphorylation, resulting in inducing the translation of uORF-tag mRNA, such as ATF4 and CHOP mRNAs. However, during stress condition, overexpression of KEPI decreased eIF2α phosphorylation, resulting in reducing the GFP reporter and CHOP proteins. This is the first report to demonstrate that KEPI plays a negative role in uORFchop - mediated translation during ER stress.

Comprehensive Detection of Pseudogenes Transcribed by Readthrough.

Transcription termination is a critical stage for the production of legitimate mRNAs, and consequently functional proteins. However, the transcription machinery can ignore the stop signs and continue elongating beyond gene boundaries, invading downstream neighboring genes. Such phenomenon, designated transcription readthrough, can trigger the expression of pseudogenes usually silenced or lacking the proper regulatory signals. Due to the sequence similarity to parental genes, readthrough transcribed pseudogenes can regulate relevant protein-coding genes and impact biological functions. Here, we describe a computational pipeline that employs already existent bioinformatic tools to detect readthrough transcribed pseudogenes from expression profiles. We also unveil that combining strand-specific transcriptome data and epigenetic profiles can enhance and corroborate the results. By applying such approach to renal cancer biopsies, we show that pseudogenes can be readthrough transcribed as part of unspliced transcripts or processed RNA chimeras. Overall, our pipeline allows us to scrutinize transcriptome profiles to detect a diversity of readthrough events leading to expression of pseudogenes.

Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae.

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3'-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5' of the stop codon, six nucleotides 3' of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3'-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after 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. Additionally, we found low readthrough genes to have shorter 3'-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

Structural basis of translation termination, rescue, and recycling in mammalian mitochondria.

The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in the course of evolution. Here, we use single-particle cryo-electron microscopy (cryo-EM) as a screening tool to identify mitochondrial translation termination mechanisms and to describe them in molecular detail. We show how mitochondrial release factor 1a releases the nascent chain from the ribosome when it encounters the canonical stop codons UAA and UAG. Furthermore, we define how the peptidyl-tRNA hydrolase ICT1 acts as a rescue factor on mitoribosomes that have stalled on truncated messages to recover them for protein synthesis. Finally, we present structural models detailing the process of mitochondrial ribosome recycling to explain how a dedicated elongation factor, mitochondrial EFG2 (mtEFG2), has specialized for cooperation with the mitochondrial ribosome recycling factor to dissociate the mitoribosomal subunits at the end of the translation process.

Defects in translation-dependent quality control pathways lead to convergent molecular and neurodevelopmental pathology.

Translation-dependent quality control pathways such as no-go decay (NGD), non-stop decay (NSD), and nonsense-mediated decay (NMD) govern protein synthesis and proteostasis by resolving non-translating ribosomes and preventing the production of potentially toxic peptides derived from faulty and aberrant mRNAs. However, how translation is altered and the in vivo defects that arise in the absence of these pathways are poorly understood. Here, we show that the NGD/NSD factors Pelo and Hbs1l are critical in mice for cerebellar neurogenesis but expendable for survival of these neurons after development. Analysis of mutant mouse embryonic fibroblasts revealed translational pauses, alteration of signaling pathways, and translational reprogramming. Similar effects on signaling pathways, including mTOR activation, the translatome and mouse cerebellar development were observed upon deletion of the NMD factor Upf2. Our data reveal that these quality control pathways that function to mitigate errors at distinct steps in translation can evoke similar cellular responses.

Live-cell imaging reveals kinetic determinants of quality control triggered by ribosome stalling.

Translation of problematic mRNA sequences induces ribosome stalling, triggering quality-control events, including ribosome rescue and nascent polypeptide degradation. To define the timing and regulation of these processes, we developed a SunTag-based reporter to monitor translation of a problematic sequence (poly[A]) in real time on single mRNAs. Although poly(A)-containing mRNAs undergo continuous translation over the timescale of minutes to hours, ribosome load is increased by ∼3-fold compared to a control, reflecting long queues of ribosomes extending far upstream of the stall. We monitor the resolution of these queues in real time and find that ribosome rescue is very slow compared to both elongation and termination. Modulation of pause strength, collision frequency, and the collision sensor ZNF598 reveals how the dynamics of ribosome collisions and their recognition facilitate selective targeting for quality control. Our results establish that slow clearance of stalled ribosomes allows cells to distinguish between transient and deleterious stalls.

Poly(A)-Binding Protein Regulates the Efficiency of Translation Termination.

Multiple factors influence translation termination efficiency, including nonsense codon identity and immediate context. To determine whether the relative position of a nonsense codon within an open reading frame (ORF) influences termination efficiency, we quantitate the production of prematurely terminated and/or readthrough polypeptides from 26 nonsense alleles of 3 genes expressed in yeast. The accumulation of premature termination products and the extent of readthrough for the respective premature termination codons (PTCs) manifest a marked dependence on PTC proximity to the mRNA 3' end. Premature termination products increase in relative abundance, whereas readthrough efficiencies decrease progressively across different ORFs, and readthrough efficiencies for a PTC increase in response to 3' UTR lengthening. These effects are eliminated and overall translation termination efficiency decreases considerably in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and also suggest that inefficient termination is a trigger for nonsense-mediated mRNA decay (NMD).

[The Influence of A/G Composition of 3' Stop Codon Contexts on Translation Termination Efficiency in Eukaryotes].

Translation termination is a finishing step of protein biosynthesis. The significant role in this process belongs not only to protein factors of translation termination but also to the nearest nucleotide environment of stop codons. There are numerous descriptions of stop codons readthrough, which is due to specific nucleotide sequences behind them. However, represented data are segmental and don't explain the mechanism of the nucleotide context influence on translation termination. It is well known that stop codon UAA usage is preferential for A/T-rich genes, and UAG, UGA-for G/C-rich genes, which is related to an expression level of these genes. We investigated the connection between a frequency of nucleotides occurrence in 3' area of stop codons in the human genome and their influence on translation termination efficiency. We found that 3' context motif, which is cognate to the sequence of a stop codon, stimulates translation termination. At the same time, the nucleotide composition of 3' sequence that differs from stop codon, decreases translation termination efficiency.

CTELS: A Cell-Free System for the Analysis of Translation Termination Rate.

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3' UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a "leaky" stop codon context, which likely defines the basis of nonsense suppression.

Nucleocytoplasmic Proteomic Analysis Uncovers eRF1 and Nonsense-Mediated Decay as Modifiers of ALS/FTD C9orf72 Toxicity.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in C9orf72 (C9-HRE). While RNA and dipeptide repeats produced by C9-HRE disrupt nucleocytoplasmic transport, the proteins that become redistributed remain unknown. Here, we utilized subcellular fractionation coupled with tandem mass spectrometry and identified 126 proteins, enriched for protein translation and RNA metabolism pathways, which collectively drive a shift toward a more cytosolic proteome in C9-HRE cells. Among these was eRF1, which regulates translation termination and nonsense-mediated decay (NMD). eRF1 accumulates within elaborate nuclear envelope invaginations in patient induced pluripotent stem cell (iPSC) neurons and postmortem tissue and mediates a protective shift from protein translation to NMD-dependent mRNA degradation. Overexpression of eRF1 and the NMD driver UPF1 ameliorate C9-HRE toxicity in vivo. Our findings provide a resource for proteome-wide nucleocytoplasmic alterations across neurodegeneration-associated repeat expansion mutations and highlight eRF1 and NMD as therapeutic targets in C9orf72-associated ALS and/or FTD.

Dbp5/DDX19 between Translational Readthrough and Nonsense Mediated Decay.

The DEAD-box protein Dbp5 (human DDX19) remodels RNA-protein complexes. Dbp5 functions in ribonucleoprotein export and translation termination. Termination occurs, when the ribosome has reached a stop codon through the Dbp5 mediated delivery of the eukaryotic termination factor eRF1. eRF1 contacts eRF3 upon dissociation of Dbp5, resulting in polypeptide chain release and subsequent ribosomal subunit splitting. Mutations in DBP5 lead to stop codon readthrough, because the eRF1 and eRF3 interaction is not controlled and occurs prematurely. This identifies Dbp5/DDX19 as a possible potent drug target for nonsense suppression therapy. Neurodegenerative diseases and cancer are caused in many cases by the loss of a gene product, because its mRNA contained a premature termination codon (PTC) and is thus eliminated through the nonsense mediated decay (NMD) pathway, which is described in the second half of this review. We discuss translation termination and NMD in the light of Dbp5/DDX19 and subsequently speculate on reducing Dbp5/DDX19 activity to allow readthrough of the PTC and production of a full-length protein to detract the RNA from NMD as a possible treatment for diseases.

First confirmatory study on PTPRQ as an autosomal dominant non-syndromic hearing loss gene.

BACKGROUND: Biallelic PTPRQ pathogenic variants have been previously reported as causative for autosomal recessive non-syndromic hearing loss. In 2018 the first heterozygous PTPRQ variant has been implicated in the development of autosomal dominant non-syndromic hearing loss (ADNSHL) in a German family. The study presented the only, so far known, PTPRQ pathogenic variant (c.6881G>A) in ADNSHL. It is located in the last PTPRQ coding exon and introduces a premature stop codon (p.Trp2294*). METHODS: A five-generation Polish family with ADNSHL was recruited for the study (n = 14). Thorough audiological, neurotological and imaging studies were carried out to precisely define the phenotype. Genomic DNA was isolated from peripheral blood samples or buccal swabs of available family members. Clinical exome sequencing was conducted for the proband. Family segregation analysis of the identified variants was performed using Sanger sequencing. Single nucleotide polymorphism array on DNA samples from the Polish and the original German family was used for genome-wide linkage analysis. RESULTS: Combining clinical exome sequencing and family segregation analysis, we have identified the same (NM_001145026.2:c.6881G>A, NP_001138498.1:p.Trp2294*) PTPRQ alteration in the Polish ADNSHL family. Using genome-wide linkage analysis, we found that the studied family and the original German family derive from a common ancestor. Deep phenotyping of the affected individuals showed that in contrast to the recessive form, the PTPRQ-related ADNSHL is not associated with vestibular dysfunction. In both families ADNSHL was progressive, affected mainly high frequencies and had a variable age of onset. CONCLUSION: Our data provide the first confirmation of PTPRQ involvement in ADNSHL. The finding strongly reinforces the inclusion of PTPRQ to the small set of genes leading to both autosomal recessive and dominant hearing loss.

uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3.

Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.

Hcr1/eIF3j Is a 60S Ribosomal Subunit Recycling Accessory Factor In Vivo.

Hcr1/eIF3j is a sub-stoichiometric subunit of eukaryotic initiation factor 3 (eIF3) that can dissociate the post-termination 40S ribosomal subunit from mRNA in vitro. We examine this ribosome recycling role in vivo by ribosome profiling and reporter assays and find that loss of Hcr1 leads to reinitiation of translation in 3' UTRs, consistent with a defect in recycling. However, the defect appears to be in the recycling of the 60S subunit, rather than the 40S subunit, because reinitiation does not require an AUG codon and is suppressed by overexpression of the 60S dissociation factor Rli1/ABCE1. Consistent with a 60S recycling role, overexpression of Hcr1 cannot compensate for loss of 40S recycling factors Tma64/eIF2D and Tma20/MCT-1. Intriguingly, loss of Hcr1 triggers greater expression of RLI1 via an apparent feedback loop. These findings suggest Hcr1/eIF3j is recruited to ribosomes at stop codons and may coordinate the transition to a new round of translation.

The antimalarial drug mefloquine enhances TP53 premature termination codon readthrough by aminoglycoside G418.

Nonsense mutations constitute ~10% of TP53 mutations in cancer. They introduce a premature termination codon that gives rise to truncated p53 protein with impaired function. The aminoglycoside G418 can induce TP53 premature termination codon readthrough and thus increase cellular levels of full-length protein. Small molecule phthalimide derivatives that can enhance the readthrough activity of G418 have also been described. To determine whether readthrough enhancers exist among drugs that are already approved for use in humans, we tested seven antimalarial drugs for readthrough of the common R213X TP53 nonsense mutation in HDQ-P1 breast cancer cells. Mefloquine induced no TP53 readthrough activity as a single agent but it strongly potentiated readthrough by G418. The two enantiomers composing pharmaceutical mefloquine potentiated readthrough to similar levels in HDQ-P1 cells and also in SW900, NCI-H1688 and HCC1937 cancer cells with different TP53 nonsense mutations. Exposure to G418 and mefloquine increased p53 phosphorylation at Ser15 and P21 transcript levels following DNA damage, indicating p53 produced via readthrough was functional. Mefloquine does not appear to enhance readthrough via lysosomotropic effects as it did not significantly affect lysosomal pH, the cellular levels of G418 or its distribution in organellar or cytosolic fractions. The availability of a readthrough enhancer that is already approved for use in humans should facilitate study of the therapeutic potential of TP53 readthrough in preclinical cancer models.

Targeting Translation Termination Machinery with Antisense Oligonucleotides for Diseases Caused by Nonsense Mutations.

Efforts to develop treatments for diseases caused by nonsense mutations have focused on identification of small molecules that promote translational read-through of messenger RNAs (mRNAs) harboring nonsense stop codons to produce full-length proteins. However, to date, no small molecule read-through drug has received FDA approval, probably because of a lack of balance between efficacy and safety. Depletion of translation termination factors eukaryotic release factor (eRF) 1 and eRF3a in cells was shown to promote translational read-through of a luciferase reporter gene harboring a nonsense mutation. In this study, we identified antisense oligonucleotides (ASOs) targeting translation termination factors and determined if ASO-mediated depletion of these factors could be a potentially effective and safe therapeutic approach for diseases caused by nonsense mutations. We found that ASO-mediated reduction of either eRF1 or eRF3a to 30%-40% of normal levels in the mouse liver is well tolerated. Hemophilia mice that express a mutant allele of human coagulation factor IX (FIX) containing nonsense mutation R338X were treated with eRF1- or eRF3a-ASO. We found that although eRF1- or eRF3a-ASO alone only elicited a moderate read-through effect on hFIX-R338X mRNA, both worked in synergy with geneticin, a small molecule read-through drug, demonstrating significantly increased production of functional full-length hFIX protein to levels that would rescue disease phenotypes in these mice. Overall our results indicate that modulating the translation termination pathway in the liver by ASOs may provide a novel approach to improving the efficacy of small molecule read-through drugs to treat human genetic diseases caused by nonsense mutations.

The aminoglycoside geneticin permits translational readthrough of the CTNS W138X nonsense mutation in fibroblasts from patients with nephropathic cystinosis.

BACKGROUND: Cystinosis is an ultrarare disorder caused by mutations of the cystinosin (CTNS) gene, encoding a cystine-selective efflux channel in the lysosomes of all cells of the body. Oral therapy with cysteamine reduces intralysosomal cystine accumulation and slows organ deterioration but cannot reverse renal Fanconi syndrome nor prevent the eventual need for renal transplantation. A definitive therapeutic remains elusive. About 15% of cystinosis patients worldwide carry one or more nonsense mutations that halt translation of the CTNS protein. Aminoglycosides such as geneticin (G418) can bind to the mammalian ribosome, relax translational fidelity, and permit readthrough of premature termination codons to produce full-length protein. METHODS: To ascertain whether aminoglycosides permit readthrough of the most common CTNS nonsense mutation, W138X, we studied the effect of G418 on patient fibroblasts. RESULTS: G418 treatment induced translational readthrough of CTNSW138X constructs transfected into HEK293 cells and expression of full-length endogenous CTNS protein in homozygous W138X fibroblasts. CONCLUSIONS: Reduction in intracellular cystine indicates that the CTNS protein produced is functional as a cystine transporter. Interestingly, similar effects were seen even in W138X compound heterozygotes. These studies establish proof-of-principle for the potential of aminoglycosides to treat cystinosis and possibly other monogenic diseases caused by nonsense mutations.