Selected Publications
Articles Related to Nonsense Suppression
A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion
Sharma, J., Du, M., Wong, Eric, Mutyam, V., Li, Y., Chen, J., Wangen, J., Thrasher, K., Fu, L., Peng, N., Tang, L., Liu, K., Mathew, B., Bostwick, R., Augelli-Szafran, C., Bihler, H., Liang, F., Mahiou, J., Saltz, J., Rab, A., Hong, J., Sorscher, E., Menhenhall, E. Coppola, C., Keeling, K., Green, R., Mense, M., Suto, M., Rowe, S., Bedwell, D.
Nature Communications 12:4358 (2021)
This study describes the discovery and characterization of a new class of readthrough compound that targets the termination factor eRF1 for proteasomal degradation.
Xue, X., Mutyam, V., Thakerar, A., Mobley, J., Bridges, R.J., Rowe, S.M., Keeling, K.M. and Bedwell, D.M. (2017) Human Molecular Genetics 26: 3116-3129.
This study investigates the mechanism behind suppressing termination at nonsense mutations found in cystic fibrosis patients. A reporter was used to facilitate purification of peptides generated from suppression of premature termination codons (PTCs). The identity of the amino acids incorporated at the PTCs during readthrough was determined by mass spectrometry. Three important results were found in this study. First, only a subset of near-cognate amino acids was incorporated at each PTC. Second, the amino acid that became incorporated at each PTC affected protein maturation and function. Third, the local mRNA sequence surrounding the PTCs affected amino acid incorporation during PTC suppression.
Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression.
Roy, B., Friesen, W., Tomizawa, Y., Leszyk, J.D., Zhou, Johnson, J.B., J. Dakka, J., Trotta, C., Xue, X., Mutyam, V., Keeling, K., Mobley, J., Rowe, S.M., Welch, E.M., Bedwell, D.M. and Jacobson, A. (2016) Proceedings of the National Academy of Sciences, USA, 113:12508-12513
This study examined the mechanism by which the small-molecule drug, ataluren, promotes suppression of premature termination codons (PTCs). Results indicate that the ribosome is likely the target for ataluren, which suppresses termination by promoting the insertion of an amino acids that are carried by tRNAs that are near-cognate to the PTC.
Discovery of clinically approved agents that promote nonsense suppression.
Mutyam, V., Du, M., Xue, X., White, E.L, Bostwick, R., Rasmussen, L., Liu, B., Mazur, M., Hong, J.S., Falk Libby, E., Liang, F., Shang, H., Mense, M., Suto, M.J., Bedwell, D.M. and Rowe, S.M. (2016) American Journal of Respiratory and Critical Care Medicine 194:1092-1103.
A library of 1,600 clinically approved compounds were screened in Fischer rat thyroid (FRT) cells expressing a dual luciferase reporter to identify molecules that suppress termination at premature termination codons (PTCs). Eight compounds were advanced to secondary screening to determine if they could suppress CFTR nonsense mutations and restore CFTR protein function. Among these, the herbal agent, escin, demonstrated PTC suppression activity and enhanced CFTR expression and function.
Long-term nonsense suppression therapy moderates MPS I-H disease.
Gunn, G., Dai, Y., Du, M., Belakhov, V., Kandeasamy, J., Schoeb, T., Baasov, T., Bedwell, D. and Keeling, K. (2014) Molecular Genetics and Metabolism 111: 374-381.
In this study, we examined the ability of the designer aminoglycoside, NB84, to suppress the Idua-W402X nonsense mutation in a mouse model of mucopolysaccharidosis I-Hurler, the most severe form of alpha-L-iduronidase deficiency. NB84 is less toxic and more efficient at suppressing nonsense mutations than traditional aminoglycosides. In a long-term, 28-week study, we found that NB84 restored alpha-L-iduronidase in multiple tissues of Idua-W402X mice, which led to corresponding reductions in glycosaminoglycan accumulation. We also found that mice treated with NB84 for 28 weeks showed improved heart morphology and function, ameliorated bone morphology, and reduced neuroimflammation. This was the first report to show that nonsense suppression therapy could attenuate onset of a disease in vivo.
Attenuation of nonsense-mediated mRNA decay enhances in vivo nonsense suppression.
Keeling, K., Wang, D., Du, M., Dai, Y., Murugesan, S., Chenna, B., Clark, J.; Belakhov, V., Kandasamy, J., Velu, S., Baasov, T., and Bedwell, D. (2013) PLoS One 8: e60478 [one of the top 10% most cited PLoS One articles as of June 2017]
In this study, we test whether inhibition of the nonsense-mediated mRNA decay (NMD) pathway will enhance suppression of a premature termination codon (PTC) in vivo. We administered the NMD inhibitor molecule, NMDI-1, to Idua-W402X mice in combination with compounds that suppressed termination at premature termination codons (PTCs). We found that inhibiting NMD did increase the amount of protein function restored by PTC suppression compared to suppression alone, suggesting that attenuating NMD efficiency is a way to enhance the efficiency of PTC suppression.
Wang, D., Belakhov, V., Kandasamy, J., Baasov, T., Li, S.-C., Li, T.-Y., Bedwell, D. and Keeling, K. (2012) Molecular Genetics and Metabolism 105: 116-125 (highlighted on cover).
In this study, a number of aminoglycoside derivatives that were designed to be less toxic and more effective at suppressing nonsense mutations were examined for their ability to suppress the Idua-W402X nonsense mutation in a mouse model of MPS I-H. Among the compounds tested, we found that NB84 mediated the most efficient suppression of the Idua-W402X mutation in both in vitro and in vivo MPS I-H models.
Du, M., Liu, X., Welch, E., Peltz, S. and Bedwell, D. (2008) Proceedings of the National Academy of Sciences, USA105: 2064-2069.
This study showed that the non-toxic nonsense suppression molecule, PTC124 (ataluren), restored CFTR function in a cystic fibrosis mouse model expressing a CFTR-G542X nonsense mutation. PTC124 restored CFTR expression in the apical surface of intestinal glands of treated mice. Furthermore, PTC124-treated mice showed a significant increase in cAMP-stimulated transepithelial chloride currents, consistent with restoration of CFTR function.
Clancy, J., Beböck, Z., Ruiz, F., King, C., Jones, J., Walker, L., Greer, H., Hong, J., Wing, L., Macaluso, M., Lyrene, R., Sorscher, E., and Bedwell, D. (2001). American Journal of Respiratory and Critical Care Medicine163: 1683-1692.
In this pilot clinical trial, cystic fibrosis patients who carry a nonsense mutation were administered the aminoglycoside gentamicin parenterally for 1 week. Patients then underwent repeated in vivo measures of CFTR activity via nasal potential difference and sweat chloride testing. Results showed that in most patients, an increase in CFTR activity was observed with gentamicin.
Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line.
Bedwell, D., Kaenjak, A., Benos, D., Bebok, Z, Clancy, J., Hong, J., Tousson, A., Bubien, J., and Sorscher, E. (1997) Nature Medicine 3: 1280-1284.
In this study, the aminoglycoside, gentamicin, was shown to suppress termination at several nonsense mutations found in cystic fibrosis (CF) patients. In addition, CFTR function and mRNA levels were found to be partially restored in a CF bronchial epithelial cell line carrying the CFTR-W1282X mutation when treated with gentamicin.
Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations.
Howard, M., Frizzell, R. and Bedwell, D. (1996) Nature Medicine 2: 467-469.
In this seminal study, the aminoglycoside gentamicin was first reported to suppress nonsense mutations associated with cystic fibrosis and restore CFTR protein function.
Articles Related to Translation Termination
Identification of eRF1 residues that play critical roles in stop codon recognition.
Conard, S., Buckley, J., Dang, M., Bedwell, G., Carter, R., Khass, M. and Bedwell, D. (2012) RNA 18: 1210-1221.
This study explores the TASNIKS and YCF motifs in domain 1 of the release factor, eRF1. These motifs are thought to play a major role in stop codon recognition. Our study found that the YCF motif appears to play a larger role in stop codon selectivity than the TASNIKS motif.
Fan-Minogue, H., Du, M., Pisarev, A., Kallmeyer, A., Salas-Marco, J., Keeling, K., Thompson, S., Pestova, T. and Bedwell, D. (2008) Molecular Cell 30: 599-609.
In this study, hybrid eRF1 proteins were generated that carried a domain 1 from Euplotes octocarinatus, a variant code organism that recognizes only UAA and UAG as stop codons, fused to domains 2 and 3 from Saccharomyces cerevisiae, a standard code organism that recognizes UAA, UAG, and UGA as stop codons. This hybrid eRF1 was mutagenized to identify mutations that restored UGA stop codon recognition. Results from this study suggest that together, the TASNIKS motif in eRF1 and eRF3 function to trigger conformational changes in eRF1 that couple stop codon recognition to peptide release.
Fan-Minogue, H. and Bedwell, D. (2007) RNA 14: 148-157.
Aminoglycoside antibiotics bind to a region of the small ribosomal subunit called the decoding center. Differences between the prokaryotic and eukaryotic decoding centers alters aminoglycoside binding. In this study, mutations were introduced into the eukaryotic decoding center to mimic the bacterial decoding center. We found that some residues in the eukaryotic decoding center were essential for viability. Others contributed to aminoglycoside resistance as well as translational misreading in the presence of aminoglycosides. These results increase our understanding of functional differences between the eukaryotic and prokaryotic decoding sites.
Keeling, K., Salas-Marco, J., Osherovich, L. and Bedwell, D. (2006) Molecular and Cellular Biology 26: 5237-5248.
We found that a previously uncharacterized protein, Tpa1p, appears to couple translation termination with mRNA stability through interactions with the termination complex and poly(A) binding protein.
Kallmeyer, A., Keeling, K. and Bedwell, D. (2006) Eukaryotic Cell 5: 1378-87.
In this study, we demonstrate that eRF1 is phosphorylated in Saccharomyces cerevisiae at two serine residues by the CK2 protein kinase. The phosphorylation status did not affect the interaction of eRF1 with eRF3 and did not alter eRF1 function in termination or affect nonsense-mediated mRNA. However, eRF1 phosphorylation did depend on carbon source availability, suggesting that phosphorylation of eRF1 may be important during cell stress.
Distinct paths to stop codon reassignment in the ciliates Tetrahymena and Euplotes.
Salas-Marco, J., Kallmeyer, A., Fan, H., Klobutcher, L., Farabaugh, P., and Bedwell, D. (2006) Molecular and Cellular Biology 26: 438-447.
Among ciliate species, reassignment of stop codons to sense codons is common. For example, in Euplotes, only UAA and UAG are used as stop codons. In Tetrahymena, only UGA is recognized as a stop codon. In this study, we utilized hybrid eRF1 proteins composed of domain 1 from either Euplotes or Tetrahymena fused to domains 2 and 3 from Saccharomyces cerevisiae to better understand how eRF1 mediates termination. Our results suggest that although domain 1 plays an important role in stop codon recognition, other factors also influence the process.
Salas-Marco, J. and Bedwell, D. (2005) Journal of Molecular Biology 348: 801-815.
Suppression of termination can be caused by reduced accuracy of translation elongation or inefficient translation termination. This study examined ways to differentiate defects in elongation from termination defects using a misincorporation reporter in Saccharamyces cerevisiae. This reporter was used to show that altering different regions of the ribosome influences elongation and termination efficiency to different extents.
GTP hydrolysis by eRF3 facilitates stop codon decoding during eukaryotic translation termination.
Salas-Marco, J. and Bedwell, D. (2004) Molecular Cell Biology 24: 7769-7778.
This study examined how the GTPase function of eRF3 affects stop codon release by eRF1 in Saccharomyces cerevisiae. Results showed that the GTPase activity of eRF3 is required to couple stop codon recognition by eRF1 to efficiency peptide release.
