Journal articles: 'Stop codon read-through'

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Crawford, Daniel K., Iris Alroy, Neal Sharpe, Matthew M. Goddeeris, and Greg Williams. "ELX-02 Generates Protein via Premature Stop Codon Read-Through without Inducing Native Stop Codon Read-Through Proteins." Journal of Pharmacology and Experimental Therapeutics 374, no. 2 (May 6, 2020): 264–72. http://dx.doi.org/10.1124/jpet.120.265595.

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Kramarski, Lior, and Eyal Arbely. "Translational read-through promotes aggregation and shapes stop codon identity." Nucleic Acids Research 48, no. 7 (March 4, 2020): 3747–60. http://dx.doi.org/10.1093/nar/gkaa136.

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Abstract Faithful translation of genetic information depends on the ability of the translational machinery to decode stop codons as termination signals. Although termination of protein synthesis is highly efficient, errors in decoding of stop codons may lead to the synthesis of C-terminally extended proteins. It was found that in eukaryotes such elongated proteins do not accumulate in cells. However, the mechanism for sequestration of C-terminally extended proteins is still unknown. Here we show that 3′-UTR-encoded polypeptides promote aggregation of the C-terminally extended proteins, and targeting to lysosomes. We demonstrate that 3′-UTR-encoded polypeptides can promote different levels of protein aggregation, similar to random sequences. We also show that aggregation of endogenous proteins can be induced by aminoglycoside antibiotics that promote stop codon read-through, by UAG suppressor tRNA, or by knokcdown of release factor 1. Furthermore, we find correlation between the fidelity of termination signals, and the predicted propensity of downstream 3′-UTR-encoded polypeptides to form intrinsically disordered regions. Our data highlight a new quality control mechanism for elimination of C-terminally elongated proteins.

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Buck, Nicole E., Leonie Wood, Ruimei Hu, and Heidi L. Peters. "Stop codon read-through of a Methylmalonic aciduria mutation." Molecular Genetics and Metabolism 97, no. 4 (August 2009): 244–49. http://dx.doi.org/10.1016/j.ymgme.2009.04.004.

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Jaafar, Fauziah Mohd, Houssam Attoui, Philippe de Micco, and Xavier de Lamballerie. "Termination and read-through proteins encoded by genome segment 9 of Colorado tick fever virus." Journal of General Virology 85, no. 8 (August 1, 2004): 2237–44. http://dx.doi.org/10.1099/vir.0.80019-0.

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Genome segment 9 (Seg-9) of Colorado tick fever virus (CTFV) is 1884 bp long and contains a large open reading frame (ORF; 1845 nt in length overall), although a single in-frame stop codon (at nt 1052–1054) reduces the ORF coding capacity by approximately 40 %. However, analyses of highly conserved RNA sequences in the vicinity of the stop codon indicate that it belongs to a class of ‘leaky terminators’. The third nucleotide positions in codons situated both before and after the stop codon, shows the highest variability, suggesting that both regions are translated during virus replication. This also suggests that the stop signal is functionally leaky, allowing read-through translation to occur. Indeed, both the truncated ‘termination’ protein and the full-length ‘read-through’ protein (VP9 and VP9′, respectively) were detected in CTFV-infected cells, in cells transfected with a plasmid expressing only Seg-9 protein products, and in the in vitro translation products from undenatured Seg-9 ssRNA. The ratios of full-length and truncated proteins generated suggest that read-through may be down-regulated by other viral proteins. Western blot analysis of infected cells and purified CTFV showed that VP9 is a structural component of the virion, while VP9′ is a non-structural protein.

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Shalev, Moran, and Timor Baasov. "When proteins start to make sense: fine-tuning of aminoglycosides for PTC suppression therapy." MedChemComm 5, no. 8 (2014): 1092–105. http://dx.doi.org/10.1039/c4md00081a.

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Manjunath, Lekha E., Anumeha Singh, Sarthak Sahoo, Ashutosh Mishra, Jinsha Padmarajan, Chaithanya G. Basavaraju, and Sandeep M. Eswarappa. "Stop codon read-through of mammalian MTCH2 leading to an unstable isoform regulates mitochondrial membrane potential." Journal of Biological Chemistry 295, no. 50 (October 7, 2020): 17009–26. http://dx.doi.org/10.1074/jbc.ra120.014253.

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Stop codon read-through (SCR) is a process of continuation of translation beyond a stop codon. This phenomenon, which occurs only in certain mRNAs under specific conditions, leads to a longer isoform with properties different from that of the canonical isoform. MTCH2, which encodes a mitochondrial protein that regulates mitochondrial metabolism, was selected as a potential read-through candidate based on evolutionary conservation observed in the proximal region of its 3′ UTR. Here, we demonstrate translational read-through across two evolutionarily conserved, in-frame stop codons of MTCH2 using luminescence- and fluorescence-based assays, and by analyzing ribosome-profiling and mass spectrometry (MS) data. This phenomenon generates two isoforms, MTCH2x and MTCH2xx (single- and double-SCR products, respectively), in addition to the canonical isoform MTCH2, from the same mRNA. Our experiments revealed that a cis-acting 12-nucleotide sequence in the proximal 3′ UTR of MTCH2 is the necessary signal for SCR. Functional characterization showed that MTCH2 and MTCH2x were localized to mitochondria with a long t1/2 (>36 h). However, MTCH2xx was found predominantly in the cytoplasm. This mislocalization and its unique C terminus led to increased degradation, as shown by greatly reduced t1/2 (<1 h). MTCH2 read-through–deficient cells, generated using CRISPR-Cas9, showed increased MTCH2 expression and, consistent with this, decreased mitochondrial membrane potential. Thus, double-SCR of MTCH2 regulates its own expression levels contributing toward the maintenance of normal mitochondrial membrane potential.

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Xu, Lijun, Yanrong Hao, Cui Li, Quan Shen, Baofeng Chai, Wei Wang, and Aihua Liang. "Identification of amino acids responsible for stop codon recognition for polypeptide chain release factor." Biochemistry and Cell Biology 91, no. 3 (June 2013): 155–64. http://dx.doi.org/10.1139/bcb-2012-0091.

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One factor involved in eukaryotic translation termination is class 1 release factor in eukaryotes (eRF1), which functions to decode stop codons. Variant code species, such as ciliates, frequently exhibit altered stop codon recognition. Studies revealed that some class-specific residues in the eRF1 N-terminal domain are responsible for stop codon reassignment in ciliates. Here, we investigated the effects on stop codon recognition of chimeric eRF1s containing the N-terminal domain of Euplotes octocarinatus and Blepharisma japonicum eRF1 fused to Saccharomyces cerevisiae M and C domains using dual luciferase read-through assays. Mutation of class-specific residues in different eRF1 classes was also studied to identify key residues and motifs involved in stop codon decoding. As expected, our results demonstrate that 3 pockets within the eRF1 N-terminal domain were involved in decoding stop codon nucleotides. However, allocation of residues to each pocket was revalued. Our data suggest that hydrophobic and class-specific surface residues participate in different functions: modulation of pocket conformation and interaction with stop codon nucleotides, respectively. Residues conserved across all eRF1s determine the relative orientation of the 3 pockets according to stop codon nucleotides. However, quantitative analysis of variant ciliate and yeast eRF1 point mutants did not reveal any correlation between evolutionary conservation of class-specific residues and termination-related functional specificity and was limited in elucidating a detailed mechanism for ciliate stop codon reassignment. Thus, based on isolation of suppressor tRNAs from Euplotes and Tetrahymena, we propose that stop codon reassignment in ciliates may be controlled by cooperation between eRF1 and suppressor tRNAs.

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Brooks, Doug A., Viv J. Muller, and John J. Hopwood. "Stop-codon read-through for patients affected by a lysosomal storage disorder." Trends in Molecular Medicine 12, no. 8 (August 2006): 367–73. http://dx.doi.org/10.1016/j.molmed.2006.06.001.

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Bradley, Michael E., Sviatoslav Bagriantsev, Namitha Vishveshwara, and Susan W. Liebman. "Guanidine reduces stop codon read-through caused by missense mutations inSUP35 orSUP45." Yeast 20, no. 7 (2003): 625–32. http://dx.doi.org/10.1002/yea.985.

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Ogawa, Atsushi, Masayoshi Hayami, Shinsuke Sando, and Yasuhiro Aoyama. "A Concept for Selection of Codon-Suppressor tRNAs Based on Read-Through Ribosome Display in anIn VitroCompartmentalized Cell-Free Translation System." Journal of Nucleic Acids 2012 (2012): 1–7. http://dx.doi.org/10.1155/2012/538129.

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Here is presented a concept forin vitroselection of suppressor tRNAs. It uses a pool of dsDNA templates in compartmentalized water-in-oil micelles. The template contains a transcription/translation trigger, an amber stop codon, and another transcription trigger for the anticodon- or anticodon loop-randomized gene for tRNASer. Upon transcription are generated two types of RNAs, a tRNA and a translatable mRNA (mRNA-tRNA). When the tRNA suppresses the stop codon (UAG) of the mRNA, the full-length protein obtained upon translation remains attached to the mRNA (read-through ribosome display) that contains the sequence of the tRNA. In this way, the active suppressor tRNAs can be selected (amplified) and their sequences read out. The enriched anticodon (CUA) was complementary to the UAG stop codon and the enriched anticodon-loop was the same as that in the natural tRNASer.

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11

Kato, Yusuke. "Translational Control using an Expanded Genetic Code." International Journal of Molecular Sciences 20, no. 4 (February 18, 2019): 887. http://dx.doi.org/10.3390/ijms20040887.

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A bio-orthogonal and unnatural substance, such as an unnatural amino acid (Uaa), is an ideal regulator to control target gene expression in a synthetic gene circuit. Genetic code expansion technology has achieved Uaa incorporation into ribosomal synthesized proteins in vivo at specific sites designated by UAG stop codons. This site-specific Uaa incorporation can be used as a controller of target gene expression at the translational level by conditional read-through of internal UAG stop codons. Recent advances in optimization of site-specific Uaa incorporation for translational regulation have enabled more precise control over a wide range of novel important applications, such as Uaa-auxotrophy-based biological containment, live-attenuated vaccine, and high-yield zero-leakage expression systems, in which Uaa translational control is exclusively used as an essential genetic element. This review summarizes the history and recent advance of the translational control by conditional stop codon read-through, especially focusing on the methods using the site-specific Uaa incorporation.

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Freitag, Johannes, Julia Ast, and Michael Bölker. "Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi." Nature 485, no. 7399 (May 2012): 522–25. http://dx.doi.org/10.1038/nature11051.

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Li, Chuan, and Jianzhi Zhang. "Stop-codon read-through arises largely from molecular errors and is generally nonadaptive." PLOS Genetics 15, no. 5 (May 23, 2019): e1008141. http://dx.doi.org/10.1371/journal.pgen.1008141.

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Tork, S. "The major 5' determinant in stop codon read-through involves two adjacent adenines." Nucleic Acids Research 32, no. 2 (January 21, 2004): 415–21. http://dx.doi.org/10.1093/nar/gkh201.

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Kariv, Revital, Michal Caspi, Naomi Fliss Isakov, and Rina Arbsfeld. "Treatment of Familial Adenomatous Polyposis Patients with APC Stop Codon Mutation by Read-Through." Gastroenterology 152, no. 5 (April 2017): S60. http://dx.doi.org/10.1016/s0016-5085(17)30557-7.

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Ho, Alexander T., and Laurence D. Hurst. "Effective Population Size Predicts Local Rates but Not Local Mitigation of Read-through Errors." Molecular Biology and Evolution 38, no. 1 (August 14, 2020): 244–62. http://dx.doi.org/10.1093/molbev/msaa210.

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Abstract In correctly predicting that selection efficiency is positively correlated with the effective population size (Ne), the nearly neutral theory provides a coherent understanding of between-species variation in numerous genomic parameters, including heritable error (germline mutation) rates. Does the same theory also explain variation in phenotypic error rates and in abundance of error mitigation mechanisms? Translational read-through provides a model to investigate both issues as it is common, mostly nonadaptive, and has good proxy for rate (TAA being the least leaky stop codon) and potential error mitigation via “fail-safe” 3′ additional stop codons (ASCs). Prior theory of translational read-through has suggested that when population sizes are high, weak selection for local mitigation can be effective thus predicting a positive correlation between ASC enrichment and Ne. Contra to prediction, we find that ASC enrichment is not correlated with Ne. ASC enrichment, although highly phylogenetically patchy, is, however, more common both in unicellular species and in genes expressed in unicellular modes in multicellular species. By contrast, Ne does positively correlate with TAA enrichment. These results imply that local phenotypic error rates, not local mitigation rates, are consistent with a drift barrier/nearly neutral model.

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Ng, Martin Y., Hong Li, Mikel D. Ghelfi, Yale E. Goldman, and Barry S. Cooperman. "Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms." Proceedings of the National Academy of Sciences 118, no. 2 (January 7, 2021): e2020599118. http://dx.doi.org/10.1073/pnas.2020599118.

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During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through–inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell’s protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

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NAGEL-WOLFRUM, KERSTIN, FABIAN MÖLLER, INESSA PENNER, and UWE WOLFRUM. "Translational read-through as an alternative approach for ocular gene therapy of retinal dystrophies caused by in-frame nonsense mutations." Visual Neuroscience 31, no. 4-5 (June 10, 2014): 309–16. http://dx.doi.org/10.1017/s0952523814000194.

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AbstractThe eye has become an excellent target for gene therapy, and gene augmentation therapy of inherited retinal disorders has made major progress in recent years. Nevertheless, a recent study indicated that gene augmentation intervention might not stop the progression of retinal degeneration in patients. In addition, for many genes, viral-mediated gene augmentation is currently not feasible due to gene size and limited packaging capacity of viral vectors as well as expression of various heterogeneous isoforms of the target gene. Thus, alternative gene-based strategies to stop or delay the retinal degeneration are necessary. This review focuses on an alternative pharmacologic treatment strategy based on the usage of translational read-through inducing drugs (TRIDs) such as PTC124, aminoglycoside antibiotics, and designer aminoglycosides for overreading in-frame nonsense mutations. This strategy has emerged as an option for up to 30–50% of all cases of recessive hereditary retinal dystrophies. In-frame nonsense mutations are single-nucleotide alterations within the gene coding sequence resulting in a premature stop codon. Consequently, translation of such mutated genes leads to the synthesis of truncated proteins, which are unable to fulfill their physiologic functions. In this context, application of TRIDs facilitates the recoding of the premature termination codon into a sense codon, thus restoring syntheses of full-length proteins. So far, clinical trials for non-ocular diseases have been initiated for diverse TRIDs. Although the clinical outcome is not analyzed in detail, an excellent safety profile, namely for PTC124, was clearly demonstrated. Moreover, recent data demonstrated sustained read-through efficacies of nonsense mutations causing retinal degeneration, as manifested in the human Usher syndrome. In addition, a strong retinal biocompatibility for PTC124 and designer aminoglycosides has been demonstrated. In conclusion, recent progress emphasizes the potential of TRIDs as an alternative pharmacologic treatment strategy for treating nonsense mutation-based retinal disorders.

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Shibata, Norihito, Nobumichi Ohoka, Yusuke Sugaki, Chiaki Onodera, Mizuho Inoue, Yoshiyuki Sakuraba, Daisuke Takakura, et al. "Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders." Journal of Biological Chemistry 290, no. 47 (October 6, 2015): 28428–37. http://dx.doi.org/10.1074/jbc.m115.670901.

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Csibra, E., I. Brierley, and N. Irigoyen. "Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus." Journal of Virology 88, no. 18 (July 2, 2014): 10364–76. http://dx.doi.org/10.1128/jvi.00898-14.

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Beznosková, Petra, Lucie Cuchalová, Susan Wagner, Christopher J. Shoemaker, Stanislava Gunišová, Tobias von der Haar, and Leoš Shivaya Valášek. "Translation Initiation Factors eIF3 and HCR1 Control Translation Termination and Stop Codon Read-Through in Yeast Cells." PLoS Genetics 9, no. 11 (November 21, 2013): e1003962. http://dx.doi.org/10.1371/journal.pgen.1003962.

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Bhakta, Sonali, Md Thoufic Anam Azad, and Toshifumi Tsukahara. "Genetic code restoration by artificial RNA editing of Ochre stop codon with ADAR1 deaminase." Protein Engineering, Design and Selection 31, no. 12 (December 1, 2018): 471–78. http://dx.doi.org/10.1093/protein/gzz005.

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Abstract Site directed mutagenesis is a very effective approach to recode genetic information. Proper linking of the catalytic domain of the RNA editing enzyme adenosine deaminase acting on RNA (ADAR) to an antisense guide RNA can convert specific adenosines (As) to inosines (Is), with the latter recognized as guanosines (Gs) during the translation process. Efforts have been made to engineer the deaminase domain of ADAR1 and the MS2 system to target specific A residues to restore G→A mutations. The target consisted of an ochre (TAA) stop codon, generated from the TGG codon encoding amino acid 58 (Trp) of enhanced green fluorescent protein (EGFP). This system had the ability to convert the stop codon (TAA) to a readable codon (TGG), thereby restoring fluorescence in a cellular system, as shown by JuLi fluorescence and LSM confocal microscopy. The specificity of the editing was confirmed by polymerase chain reaction-restriction fragment length polymorphism, as the restored EGFP mRNA could be cleaved into fragments of 160 and 100 base pairs. Direct sequencing analysis with both sense and antisense primers showed that the restoration rate was higher for the 5′ than for the 3′A. This system may be very useful for treating genetic diseases that result from G→A point mutations. Successful artificial editing of RNA in vivo can accelerate research in this field, and pioneer genetic code restoration therapy, including stop codon read-through therapy, for various genetic diseases.

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Gozalbo, Daniel, and Stefan Hohmann. "The naturally occurring silent invertase structural gene suc2° contains an amber stop codon that is occasionally read through." Molecular and General Genetics MGG 216, no. 2-3 (April 1989): 511–16. http://dx.doi.org/10.1007/bf00334398.

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Azimov, Rustam, Natalia Abuladze, Pakan Sassani, Debra Newman, Liyo Kao, Weixin Liu, Nicholas Orozco, Piotr Ruchala, Alexander Pushkin, and Ira Kurtz. "G418-mediated ribosomal read-through of a nonsense mutation causing autosomal recessive proximal renal tubular acidosis." American Journal of Physiology-Renal Physiology 295, no. 3 (September 2008): F633—F641. http://dx.doi.org/10.1152/ajprenal.00015.2008.

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Autosomal recessive proximal renal tubular acidosis is caused by mutations in the SLC4A4 gene encoding the electrogenic sodium bicarbonate cotransporter NBCe1-A. The mutations that have been characterized thus far result in premature truncation, mistargeting, or decreased function of the cotransporter. Despite bicarbonate treatment to correct the metabolic acidosis, extrarenal manifestations persist, including glaucoma, cataracts, corneal opacification, and mental retardation. Currently, there are no known therapeutic approaches that can specifically target mutant NBCe1-A proteins. In the present study, we tested the hypothesis that the NBCe1-A-Q29X mutation can be rescued in vitro by treatment with aminoglycoside antibiotics, which are known for their ability to suppress premature stop codons. As a model system, we cloned the NBCe1-A-Q29X mutant into a vector lacking an aminoglycoside resistance gene and transfected the mutant cotransporter in HEK293-H cells. Cells transfected with the NBCe1-A-Q29X mutant failed to express the cotransporter because of the premature stop codon. Treatment of the cells with G418 significantly increased the expression of the full-length cotransporter, as assessed by immunoblot analysis. Furthermore, immunocytochemical studies demonstrated that G418 treatment induced cotransporter expression on the plasma membrane whereas in the absence of G418, NBCe1-A-Q29X was not expressed. In HEK293-H cells transfected with the NBCe1-A-Q29X mutant not treated with G418, NBCe1-A-mediated flux was not detectable. In contrast, in cells transfected with the NBCe1-A-Q29X mutant, G418 treatment induced Na+- and HCO3−-dependent transport that did not differ from wild-type NBCe1-A function. G418 treatment in mock-transfected cells was without effect. In conclusion, G418 induces ribosomal read-through of the NBCe1-A-Q29X mutation in HEK293-H cells. These findings represent the first evidence that in the presence of the NBCe1-A-Q29X mutation that causes proximal renal tubular acidosis, full-length functional NBCe1-A protein can be produced. Our results provide the first demonstration of a mutation in NBCe1-A that has been treated in a targeted and specific manner.

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Goodenough, E., T. M. Robinson, M. B. Zook, K. M. Flanigan, J. F. Atkins, M. T. Howard, and L. C. Eisenlohr. "Cryptic MHC class I-binding peptides are revealed by aminoglycoside-induced stop codon read-through into the 3' UTR." Proceedings of the National Academy of Sciences 111, no. 15 (March 31, 2014): 5670–75. http://dx.doi.org/10.1073/pnas.1402670111.

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Herring, Christopher D., and Frederick R. Blattner. "Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator frmR." Journal of Bacteriology 186, no. 20 (October 15, 2004): 6714–20. http://dx.doi.org/10.1128/jb.186.20.6714-6720.2004.

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ABSTRACT Expression of an amber suppressor tRNA should result in read-through of the 326 open reading frames (ORFs) that terminate with amber stop codons in the Escherichia coli genome, including six pseudogenes. Abnormal extension of an ORF might alter the activities of the protein and have effects on cellular physiology, while suppression of a pseudogene could lead to a gain of function. We used oligonucleotide microarrays to determine if any effects were apparent at the level of transcription in glucose minimal medium. Surprisingly, only eight genes had significantly different expression in the presence of the suppressor. Among these were the genes yaiN, adhC, and yaiM, forming a single putative operon whose likely function is the degradation of formaldehyde. Expression of wild-type yaiN was shown to result in repression of the operon, while a suppression-mimicking allele lacking the amber stop codon and extended 7 amino acids did not. The operon was shown to be induced by formaldehyde, and the genes have been renamed frmR, frmA, and frmB, respectively.

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Long, Lu, Xudong Yang, Mark Southwood, Stephen Moore, Alexi Crosby, Paul D. Upton, Benjamin J. Dunmore, and Nicholas W. Morrell. "Targeting translational read-through of premature termination mutations in BMPR2 with PTC124 for pulmonary arterial hypertension." Pulmonary Circulation 10, no. 3 (July 2020): 204589402093578. http://dx.doi.org/10.1177/2045894020935783.

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Pulmonary arterial hypertension is a fatal disorder of the lung circulation in which accumulation of vascular cells progressively obliterates the pulmonary arterioles. This results in sustained elevation in pulmonary artery pressure leading eventually to right heart failure. Approximately, 80% of familial and 20% of sporadic idiopathic pulmonary arterial hypertension cases are caused by mutations in the bone morphogenetic protein receptor type 2 (BMPR2). Nonsense mutations in BMPR2 are amongst the most common mutations found, where the insertion of a premature termination codon causes mRNA degradation via activation of the nonsense-mediated decay pathway leading to a state of haploinsufficiency. Ataluren (PTC124), a compound that permits ribosomal read-through of premature stop codons, has been previously reported to increase BMPR2 protein expression in cells derived from pulmonary arterial hypertension patients harbouring nonsense mutations. In this study, we characterised the effects of PTC124 on a range of nonsense BMPR2 mutations, focusing on the R584X mutation both in vitro and in vivo. Treatment with PTC124 partially restored BMPR2 protein expression in blood outgrowth endothelial cells isolated from a patient harbouring the R584X mutation. Furthermore, a downstream bone morphogenetic protein signalling target, Id1, was rescued by PTC124 treatment. Mutant cells also exhibited increased lipopolysaccharide-induced permeability, which was reversed by PTC124 treatment. Increased proliferation and apoptosis in R584X blood outgrowth endothelial cells were also significantly reduced by PTC124. Moreover, oral PTC124 increased lung BMPR2 protein expression in mice harbouring the R584X mutation ( Bmpr2 +/R584X). Our findings provide support for future experimental medicine studies of PTC124 in pulmonary arterial hypertension patients with specific nonsense BMPR2 mutations.

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Wills, N. M., R. F. Gesteland, and J. F. Atkins. "Evidence that a downstream pseudoknot is required for translational read-through of the Moloney murine leukemia virus gag stop codon." Proceedings of the National Academy of Sciences 88, no. 16 (August 15, 1991): 6991–95. http://dx.doi.org/10.1073/pnas.88.16.6991.

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Shimizu-Motohashi, Yuko, Hirofumi Komaki, Norio Motohashi, Shin’ichi Takeda, Toshifumi Yokota, and Yoshitsugu Aoki. "Restoring Dystrophin Expression in Duchenne Muscular Dystrophy: Current Status of Therapeutic Approaches." Journal of Personalized Medicine 9, no. 1 (January 7, 2019): 1. http://dx.doi.org/10.3390/jpm9010001.

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Duchenne muscular dystrophy (DMD), a rare genetic disorder characterized by progressive muscle weakness, is caused by the absence or a decreased amount of the muscle cytoskeletal protein dystrophin. Currently, several therapeutic approaches to cure DMD are being investigated, which can be categorized into two groups: therapies that aim to restore dystrophin expression, and those that aim to compensate for the lack of dystrophin. Therapies that restore dystrophin expression include read-through therapy, exon skipping, vector-mediated gene therapy, and cell therapy. Of these approaches, the most advanced are the read-through and exon skipping therapies. In 2014, ataluren, a drug that can promote ribosomal read-through of mRNA containing a premature stop codon, was conditionally approved in Europe. In 2016, eteplirsen, a morpholino-based chemical capable of skipping exon 51 in premature mRNA, received conditional approval in the USA. Clinical trials on vector-mediated gene therapy carrying micro- and mini- dystrophin are underway. More innovative therapeutic approaches include CRISPR/Cas9-based genome editing and stem cell-based cell therapies. Here we review the current status of therapeutic approaches for DMD, focusing on therapeutic approaches that can restore dystrophin.

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Handy, Diane E., Yufeng Zhang, and Joseph Loscalzo. "Homocysteine Down-regulates Cellular Glutathione Peroxidase (GPx1) by Decreasing Translation." Journal of Biological Chemistry 280, no. 16 (February 25, 2005): 15518–25. http://dx.doi.org/10.1074/jbc.m501452200.

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Hyperhomocysteinemia contributes to vascular dysfunction and an increase in the risk of cardiovascular disease. An elevated level of homocysteinein vivoand in cell culture systems results in a decrease in the activity of cellular glutathione peroxidase (GPx1), an intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides. In this study, we show that homocysteine interferes with GPx1 protein expression without affecting transcript levels. Expression of the selenocysteine (SEC)-containing GPx1 protein requires special translational cofactors to “read-through” a UGA-stop codon that specifies SEC incorporation at the active site of the enzyme. These factors include a selenocysteine incorporation sequence (SECIS) in the 3′-untranslated region of the GPx1 mRNA and cofactors involved in the biosynthesis and translational insertion of SEC. To monitor SEC incorporation, we used a reporter gene system that has a UGA codon within the protein-coding region of the luciferase mRNA. Addition of either the GPx1 or GPx3 SECIS element in the 3′-untranslated region of the luciferase gene stimulated read-through by 6–11-fold in selenium-replete cells; absence of selenium prevented translation. To alter cellular homocysteine production, we used methionine in the presence of aminopterin, a folate antagonist, co-administered with hypoxanthine and thymidine (HAT/Met). This treatment increased homocysteine levels in the media by 30% (p< 0.01) and decreased GPx1 enzyme activity by 45% (p= 0.0028). HAT/Met treatment decreased selenium-mediated read-through significantly (p< 0.001) in luciferase constructs containing the GPx1 or GPx3 SECIS element; most importantly, the suppression of selenium-dependent read-through was similar whether an SV40 promoter or the GPx1 promoter was used to drive transcription of the SECIS-containing constructs. Furthermore, HAT/Met had no effect on steady-state GPx1 mRNA levels but decreased GPx1 protein levels, suggesting that this effect is not transcriptionally mediated. These data support the conclusion that homocysteine decreases GPx1 activity by altering the translational mechanism essential for the synthesis of this selenocysteine-containing protein.

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Dündar, Halil, Gürsel Biberoğlu, Aslı İnci, İpek Işık Gönül, İlyas Okur, and Leyla Tümer. "The chemical chaperone 4-phenylbutyrate enhances alpha-galactosidase activity subsequent to stop-codon read-through therapy with triamterene in Fabry R227X fibroblasts." Molecular Genetics and Metabolism 132, no. 2 (February 2021): S35—S36. http://dx.doi.org/10.1016/j.ymgme.2020.12.068.

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Aslam, Muhammad Assad, Mir Farshid Alemdehy, Bingtao Hao, Peter H. L. Krijger, Colin E. J. Pritchard, Iris de Rink, Fitriari Izzatunnisa Muhaimin, et al. "The Ig heavy chain protein but not its message controls early B cell development." Proceedings of the National Academy of Sciences 117, no. 49 (November 23, 2020): 31343–52. http://dx.doi.org/10.1073/pnas.2004810117.

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Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearrangedIghallele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productiveIghallele is expressed, a phenomenon known asIghallelic exclusion. In contrast to a productively rearrangedIghallele, theIghmessenger RNA (mRNA) (IgHR) from a nonproductively rearrangedIghallele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stableIgHRto the molecular and developmental changes associated with the IgHCC. This point was addressed by generating theIghTer5H∆TMmouse model fromIghTer5Hmice having a premature termination codon at position +5 in leader exon ofIghTer5Hallele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation ofIghTer5Hmessage, indicating that previous conclusions regarding a role ofIgHRin establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, theIghTer5H∆TMknock-in allele, which generated stableIgHRbut no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and notIgHRexpression.

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Leroux, Alena, France Leturcq, Nathalie Deburgrave, and Marie-France Szajnert. "Prenatal diagnosis of recessive congenital methaemoglobinaemia type II: novel mutation in the NADH-cytochrome b5 reductase gene leading to stop codon read-through." European Journal of Haematology 74, no. 5 (May 2005): 389–95. http://dx.doi.org/10.1111/j.1600-0609.2004.00388.x.

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Strawn, Lisa A., Changyi A. Lin, Elizabeth M. H. Tank, Morwan M. Osman, Sarah A. Simpson, and Heather L. True. "Mutants of the Paf1 Complex Alter Phenotypic Expression of the Yeast Prion [PSI+]." Molecular Biology of the Cell 20, no. 8 (April 15, 2009): 2229–41. http://dx.doi.org/10.1091/mbc.e08-08-0813.

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The yeast [PSI+] prion is an epigenetic modifier of translation termination fidelity that causes nonsense suppression. The prion [PSI+] forms when the translation termination factor Sup35p adopts a self-propagating conformation. The presence of the [PSI+] prion modulates survivability in a variety of growth conditions. Nonsense suppression is essential for many [PSI+]-mediated phenotypes, but many do not appear to be due to read-through of a single stop codon, but instead are multigenic traits. We hypothesized that other global mechanisms act in concert with [PSI+] to influence [PSI+]-mediated phenotypes. We have identified one such global regulator, the Paf1 complex (Paf1C). Paf1C is conserved in eukaryotes and has been implicated in several aspects of transcriptional and posttranscriptional regulation. Mutations in Ctr9p and other Paf1C components reduced [PSI+]-mediated nonsense suppression. The CTR9 deletion also alters nonsense suppression afforded by other genetic mutations but not always to the same extent as the effects on [PSI+]-mediated read-through. Our data suggest that the Paf1 complex influences mRNA translatability but not solely through changes in transcript stability or abundance. Finally, we demonstrate that the CTR9 deletion alters several [PSI+]-dependent phenotypes. This provides one example of how [PSI+] and genetic modifiers can interact to uncover and regulate phenotypic variability.

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Sarkar, Chinmoy, Zhongjian Zhang, and Anil B. Mukherjee. "Stop codon read-through with PTC124 induces palmitoyl-protein thioesterase-1 activity, reduces thioester load and suppresses apoptosis in cultured cells from INCL patients." Molecular Genetics and Metabolism 104, no. 3 (November 2011): 338–45. http://dx.doi.org/10.1016/j.ymgme.2011.05.021.

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Lashkevich, Kseniya A., Valeriya I. Shlyk, Artem S. Kushchenko, Vadim N. Gladyshev, Elena Z. Alkalaeva, and Sergey E. Dmitriev. "CTELS: A Cell-Free System for the Analysis of Translation Termination Rate." Biomolecules 10, no. 6 (June 16, 2020): 911. http://dx.doi.org/10.3390/biom10060911.

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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.

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Louis, E. J., and J. E. Haber. "Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors." Genetics 123, no. 1 (September 1, 1989): 81–95. http://dx.doi.org/10.1093/genetics/123.1.81.

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Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

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Wang, Bingjing, Zhaohui Yang, Becky K. Brisson, Huisheng Feng, Zhiqian Zhang, Ellen M. Welch, Stuart W. Peltz, Elisabeth R. Barton, Robert H. Brown, and H. Lee Sweeney. "Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression." Journal of Applied Physiology 109, no. 3 (September 2010): 901–5. http://dx.doi.org/10.1152/japplphysiol.01366.2009.

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Mutations that result in the loss of the protein dysferlin result in defective muscle membrane repair and cause either a form of limb girdle muscular dystrophy (type 2B) or Miyoshi myopathy. Most patients are compound heterozygotes, often carrying one allele with a nonsense mutation. Using dysferlin-deficient mouse and human myocytes, we demonstrated that membrane blebbing in skeletal muscle myotubes in response to hypotonic shock requires dysferlin. Based on this, we developed an in vitro assay to assess rescue of dysferlin function in skeletal muscle myotubes. This blebbing assay may be useful for drug discovery/validation for dysferlin deficiency. With this assay, we demonstrate that the nonsense suppression drug, ataluren (PTC124), is able to induce read-through of the premature stop codon in a patient with a R1905X mutation in dysferlin and produce sufficient functional dysferlin (∼15% of normal levels) to rescue myotube membrane blebbing. Thus ataluren is a potential therapeutic for dysferlin-deficient patients harboring nonsense mutations.

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Huang, Xuan, Mengnan Tian, Ciria C. Hernandez, Ningning Hu, and Robert L. Macdonald. "The GABRG2 nonsense mutation, Q40X, associated with Dravet syndrome activated NMD and generated a truncated subunit that was partially rescued by aminoglycoside-induced stop codon read-through." Neurobiology of Disease 48, no. 1 (October 2012): 115–23. http://dx.doi.org/10.1016/j.nbd.2012.06.013.

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Xu, Liai, Tingting Liu, Xingpeng Xiong, Weimiao Liu, Youjian Yu, and Jiashu Cao. "AtC3H18L is a stop-codon read-through gene and encodes a novel non-tandem CCCH zinc-finger protein that can form cytoplasmic foci similar to mRNP granules." Biochemical and Biophysical Research Communications 528, no. 1 (July 2020): 140–45. http://dx.doi.org/10.1016/j.bbrc.2020.05.081.

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von der Haar, Tobias, Jane E. Leadsham, Aimie Sauvadet, Daniel Tarrant, Ilectra S. Adam, Kofo Saromi, Peter Laun, et al. "The control of translational accuracy is a determinant of healthy ageing in yeast." Open Biology 7, no. 1 (January 2017): 160291. http://dx.doi.org/10.1098/rsob.160291.

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Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing.

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Zhao, Chengquan, Tung Nguyen, Lide Liu, Randy E. Sacco, Kim A. Brogden, and Robert I. Lehrer. "Gallinacin-3, an Inducible Epithelial β-Defensin in the Chicken." Infection and Immunity 69, no. 4 (April 1, 2001): 2684–91. http://dx.doi.org/10.1128/iai.69.4.2684-2691.2001.

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ABSTRACT Gallinacin-3 and gallopavin-1 (GPV-1) are newly characterized, epithelial β-defensins of the chicken (Gallus gallus) and turkey (Meleagris gallopavo), respectively. In normal chickens, the expression of gallinacin-3 was especially prominent in the tongue, bursa of Fabricius, and trachea. It also occurred in other organs, including the skin, esophagus, air sacs, large intestine, and kidney. Tracheal expression of gallinacin-3 increased significantly after experimental infection of chickens with Haemophilus paragallinarum, whereas its expression in the tongue, esophagus, and bursa of Fabricius was unaffected. The precursor of gallinacin-3 contained a long C-terminal extension not present in the prepropeptide. By comparing the cDNA sequences of gallinacin-3 and GPV-1, we concluded that a 2-nucleotide insertion into the gallinacin-3 gene had induced a frameshift that read through the original stop codon and allowed the chicken propeptide to lengthen. The striking structural resemblance of the precursors of β-defensins to those of crotamines (highly toxic peptides found in rattlesnake venom) supports their homology, even though defensins are specialized to kill microorganisms and crotamines are specialized to kill much larger prey.

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Attoui, Houssam, Corinne Sailleau, Fauziah Mohd Jaafar, Mourad Belhouchet, Philippe Biagini, Jean François Cantaloube, Philippe de Micco, Peter Mertens, and Stephan Zientara. "Complete nucleotide sequence of Middelburg virus, isolated from the spleen of a horse with severe clinical disease in Zimbabwe." Journal of General Virology 88, no. 11 (November 1, 2007): 3078–88. http://dx.doi.org/10.1099/vir.0.83076-0.

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The complete nucleotide sequence of Middelburg virus (MIDV) was determined for strain MIDV-857 from Zimbabwe. The isolation of this virus in 1993 from a horse that died showing severe clinical signs represents the first indication that MIDV can cause severe disease in equids. Full-length cDNA copies of the viral genome were successfully synthesized by an innovative RT-PCR amplification approach using an ‘anchor primer’ combined with the SMART methodology described previously for the synthesis of full-length cDNA copies from genome segments of dsRNA viruses. The MIDV-857 genome is 11 674 nt, excluding the 5′-terminal cap structure and poly(A) tail (which varies in length from approximately 180 to approximately 220 residues). The organization of the genome is like that of other alphaviruses, including a read-through stop codon between the nsP3 and nsP4 genes. However, phylogenetic analyses of the structural protein amino acid sequences suggested that the MIDV E1 gene was generated by recombination with a Semliki Forest virus-like virus. This hypothesis was supported by bootscanning analysis using a recombination-detection program. The 3′ untranslated region of MIDV-857 also contains a 112 nt duplication. This study reports the first full-length sequence of MIDV, which was obtained from a single RT-PCR product.

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Sun, Rong, Shaoyan Zhang, Limin Zheng, and Feng Qu. "Translation-Independent Roles of RNA Secondary Structures within the Replication Protein Coding Region of Turnip Crinkle Virus." Viruses 12, no. 3 (March 22, 2020): 350. http://dx.doi.org/10.3390/v12030350.

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RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication.

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Sułuja, Elzbieta, Ludmiła Strokowskaja, Włodzimierz Zagórski-Ostoja, and Andrzej Pałucha. "Virus-like particles of potato leafroll virus as potential carrier system for nucleic acids." Acta Biochimica Polonica 52, no. 3 (September 30, 2005): 699–702. http://dx.doi.org/10.18388/abp.2005_3433.

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Potato leafroll virus is a member of the polerovirus genus. The isometric virion is formed by a coat protein encapsidating single-stranded, positive-sense, mono-partite genomic RNA with covalently attached viral protein at the 5' end. The coat protein of the virus exists in two forms: i) a 23 kDa protein, the product of the coat protein gene, and ii) a 78 kDa protein, the product of the coat protein gene and an additional open reading frame expressed by read-through of the coat protein gene stop codon. The aim of this work was the expression of potato leafroll virus coat protein-based proteins that would be able to assemble into virus-like particles in insect cells. These modified particles were tested for their ability to encapsidate nucleic acids. Two types of N-terminally His-tagged coat protein constructs were used for the expression in insect cells: one, encoding a 23 kDa protein with the C-terminal amino-acid sequence corresponding to the wild type coat protein and the second with additional clathrin binding domain at the C-terminus. The expression of these two proteins by a recombinant baculovirus was characterized by Western immunoblotting with antibodies directed against potato leafroll virus. The protection or putative encapsidation of nucleic acids by these two coat protein derivatives was shown by DNase I and RNase A protection assays.

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Talsness, Dana M., Joseph J. Belanto, and James M. Ervasti. "Disease-proportional proteasomal degradation of missense dystrophins." Proceedings of the National Academy of Sciences 112, no. 40 (September 21, 2015): 12414–19. http://dx.doi.org/10.1073/pnas.1508755112.

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The 427-kDa protein dystrophin is expressed in striated muscle where it physically links the interior of muscle fibers to the extracellular matrix. A range of mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a typically milder form known as Becker (BMD). Patients with nonsense mutations in dystrophin are specifically targeted by stop codon read-through drugs, whereas out-of-frame deletions and insertions are targeted by exon-skipping therapies. Both treatment strategies are currently in clinical trials. Dystrophin missense mutations, however, cause a wide range of phenotypic severity in patients. The molecular and cellular consequences of such mutations are not well understood, and there are no therapies specifically targeting this genotype. Here, we have modeled two representative missense mutations, L54R and L172H, causing DMD and BMD, respectively, in full-length dystrophin. In vitro, the mutation associated with the mild phenotype (L172H) caused a minor decrease in tertiary stability, whereas the L54R mutation associated with a severe phenotype had a more dramatic effect. When stably expressed in mammalian muscle cells, the mutations caused steady-state decreases in dystrophin protein levels inversely proportional to the tertiary stability and directly caused by proteasomal degradation. Both proteasome inhibitors and heat shock activators were able to increase mutant dystrophin to WT levels, establishing the new cell lines as a platform to screen for potential therapeutics personalized to patients with destabilized dystrophin.

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Simmons, Zoe R., Amanda Sherwood, Selena Li, Sylvie Garneau-Tsodikova, and Matthew Gentry. "2348 Lafora disease premature termination codons (PTCs) are likely candidates for suppression by aminoglycosides." Journal of Clinical and Translational Science 2, S1 (June 2018): 16–17. http://dx.doi.org/10.1017/cts.2018.90.

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OBJECTIVES/SPECIFIC AIMS: A small molecule therapy is within reach to treat a molecular mechanism known to result in thousands of fatal diseases. For 10% of patients with a genetic disease, a nonsense/STOP mutation/premature termination codon (PTC) is the underlying cause of their malady. PTCs prematurely stop protein synthesis and yield truncated proteins. Truncated proteins typically provide little to no proper function or activity and are rapidly degraded; thus, disease is imminent. Recent work has demonstrated that small molecules including aminoglycosides can cause the ribosome to readthrough these PTCs. Thus, PTC readthrough with small molecules is a very attractive approach for treating diseases caused by PTCs. Small molecules that promote readthrough act on the ribosome and induce a ribosomal conformational change. In this conformation, the PTC is not recognized by the translational machinery and an amino acid is incorporated into the growing peptide chain, thus protein synthesis continues and does not stop. The use of a single small molecule to readthrough various PTC mutations has been repeatedly effective for in vitro studies and some of these have progressed to clinical trials. Although there has been success in defining these small molecules, the field has discovered that every PTC is unique and likely requires a different small molecule. Thus, developing a cell culture model to test read-through of Lafora PTCs and the functionality of the protein product is the first step to developing a readthrough therapy for a LD. METHODS/STUDY POPULATION: Method for in vitro quantification of readthrough: 24 hours before transfection, HEK293 cells were split in 6-well plates. On the following day, approximately 60% confluence, the cells were transiently transfected with the WT or PTC mutated constructs using Polyethylenimine HCl MAX. Cells were transfected with a total amount of 0.35 μg DNA/well and 2 μl Polyethylenimine HCl MAX/well. Four hours later, the transfection medium was removed and replaced with fresh medium, without streptomycin and penicillin. The fresh media contained gentamicin diluted to the indicated concentration per well. Fresh gentamicin-containing medium was replaced after 24 hours. After 48 hours, lysates were collected in 100 μL mRIPA supplemented with protease inhibitors for each construct. The lysates were run on a western blot and the N-terminal was probed with anti-FLAG. A malachite green phosphatase assay to measure inorganic phosphate release from phospho-glucans, that is glycogen or LBs. Glycogen is used in this laforin bioassay as the biologically relevant substrate in order to determine the specific activity of the readthrough products. All reactions are incubated for 40 minute the absorbance is measured at 620 nm and the pmoles of phosphate released/min/nmol protein was calculated using a standard curve. RESULTS/ANTICIPATED RESULTS: HEK293 cells were transfected with MeCP2 R241X, laforin R241X, or laforin WT NT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. HEK293 cells were transfected with WT laforin or a laforin PTC CT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. B. Quantification of read-through for PTC experiments. *p-value≤0.001. #p-value≤0.001. Schematic of laforin bioassay. The assay has been performed with human and mouse tissue as well as cultured cells. B. Laforin bioassay results using laforin from PTC experiment. **p-value≤0.001. *p-value≤0.01. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that gentamicin is not only responsible for inducing readthrough of the PTC mutations, but also for promoting translation of fully functional laforin. Therefore, our in vitro system for the analysis of PTC readthrough of laforin will be useful for determining which PTC mutations are suppressible with gentamicin or other small molecules, in what quantities laforin is recovered from PTC mutations, and if the protein products possess the appropriate enzymatic function.

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Kubaski, Francyne, Fabiano de Oliveira Poswar, Kristiane Michelin-Tirelli, Ursula da Silveira Matte, Dafne D. Horovitz, Anneliese Lopes Barth, Guilherme Baldo, Filippo Vairo, and Roberto Giugliani. "Mucopolysaccharidosis Type I." Diagnostics 10, no. 3 (March 16, 2020): 161. http://dx.doi.org/10.3390/diagnostics10030161.

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Mucopolysaccharidosis type I (MPS I) is caused by the deficiency of α-l-iduronidase, leading to the storage of dermatan and heparan sulfate. There is a broad phenotypical spectrum with the presence or absence of neurological impairment. The classical form is known as Hurler syndrome, the intermediate form as Hurler–Scheie, and the most attenuated form is known as Scheie syndrome. Phenotype seems to be largely influenced by genotype. Patients usually develop several somatic symptoms such as abdominal hernias, extensive dermal melanocytosis, thoracolumbar kyphosis odontoid dysplasia, arthropathy, coxa valga and genu valgum, coarse facial features, respiratory and cardiac impairment. The diagnosis is based on the quantification of α-l-iduronidase coupled with glycosaminoglycan analysis and gene sequencing. Guidelines for treatment recommend hematopoietic stem cell transplantation for young Hurler patients (usually at less than 30 months of age). Intravenous enzyme replacement is approved and is the standard of care for attenuated—Hurler–Scheie and Scheie—forms (without cognitive impairment) and for the late-diagnosed severe—Hurler—cases. Intrathecal enzyme replacement therapy is under evaluation, but it seems to be safe and effective. Other therapeutic approaches such as gene therapy, gene editing, stop codon read through, and therapy with small molecules are under development. Newborn screening is now allowing the early identification of MPS I patients, who can then be treated within their first days of life, potentially leading to a dramatic change in the disease’s progression. Supportive care is very important to improve quality of life and might include several surgeries throughout the life course.

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Chen, Chaoping, and Ronald C. Montelaro. "Characterization of RNA Elements That Regulate Gag-Pol Ribosomal Frameshifting in Equine Infectious Anemia Virus." Journal of Virology 77, no. 19 (October 1, 2003): 10280–87. http://dx.doi.org/10.1128/jvi.77.19.10280-10287.2003.

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ABSTRACT Synthesis of Gag-Pol polyproteins of retroviruses requires ribosomes to shift translational reading frame once or twice in a −1 direction to read through the stop codon in the gag reading frame. It is generally believed that a slippery sequence and a downstream RNA structure are required for the programmed −1 ribosomal frameshifting. However, the mechanism regulating the Gag-Pol frameshifting remains poorly understood. In this report, we have defined specific mRNA elements required for sufficient ribosomal frameshifting in equine anemia infectious virus (EIAV) by using full-length provirus replication and Gag/Gag-Pol expression systems. The results of these studies revealed that frameshifting efficiency and viral replication were dependent on a characteristic slippery sequence, a five-base-paired GC stretch, and a pseudoknot structure. Heterologous slippery sequences from human immunodeficiency virus type 1 and visna virus were able to substitute for the EIAV slippery sequence in supporting EIAV replication. Disruption of the GC-paired stretch abolished the frameshifting required for viral replication, and disruption of the pseudoknot reduced the frameshifting efficiency by 60%. Our data indicated that maintenance of the essential RNA signals (slippery sequences and structural elements) in this region of the genomic mRNA was critical for sufficient ribosomal frameshifting and EIAV replication, while concomitant alterations in the amino acids translated from the same region of the mRNA could be tolerated during replication. The data further indicated that proviral mutations that reduced frameshifting efficiency by as much as 50% continued to sustain viral replication and that greater reductions in frameshifting efficiency lead to replication defects. These studies define for the first time the RNA sequence and structural determinants of Gag-Pol frameshifting necessary for EIAV replication, reveal novel aspects relative to frameshifting elements described for other retroviruses, and provide new genetic determinants that can be evaluated as potential antiviral targets.

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Harrell, L. "Predominance of six different hexanucleotide recoding signals 3' of read-through stop codons." Nucleic Acids Research 30, no. 9 (May 1, 2002): 2011–17. http://dx.doi.org/10.1093/nar/30.9.2011.

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Journal articles on the topic 'Stop codon read-through'

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