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      Nonsense-mediated mRNA decay efficiency varies in choroideremia providing a target to boost small molecule therapeutics

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          Abstract

          Choroideremia (CHM) is an x-linked recessive chorioretinal dystrophy, with 30% caused by nonsense mutations in the CHM gene resulting in an in-frame premature termination codon (PTC). Nonsense-mediated mRNA decay (NMD) is the cell’s natural surveillance mechanism that detects and destroys PTC-containing transcripts, with UPF1 being the central NMD modulator. NMD efficiency can be variable amongst individuals with some transcripts escaping destruction, leading to the production of a truncated non-functional or partially functional protein. Nonsense suppression drugs, such as ataluren, target these transcripts and read-through the PTC, leading to the production of a full length functional protein. Patients with higher transcript levels are considered to respond better to these drugs, as more substrate is available for read-through. Using Quantitative reverse transcription PCR (RT-qPCR), we show that CHM mRNA expression in blood from nonsense mutation CHM patients is 2.8-fold lower than controls, and varies widely amongst patients, with 40% variation between those carrying the same UGA mutation [c.715 C>T; p.(R239 *)]. These results indicate that although NMD machinery is at work, efficiency is highly variable and not wholly dependent on mutation position. No significant difference in CHM mRNA levels was seen between two patients’ fibroblasts and their induced pluripotent stem cell-derived retinal pigment epithelium. There was no correlation between CHM mRNA expression and genotype, phenotype or UPF1 transcript levels. NMD inhibition with caffeine was shown to restore CHM mRNA transcripts to near wild-type levels. Baseline mRNA levels may provide a prognostic indicator for response to nonsense suppression therapy, and caffeine may be a useful adjunct to enhance treatment efficacy where indicated.

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          Most cited references18

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          The rules and impact of nonsense-mediated mRNA decay in human cancers

          Premature termination codons (PTCs) cause a large proportion of inherited human genetic diseases. PTC-containing transcripts can be degraded by an mRNA surveillance pathway termed nonsense-mediated mRNA decay (NMD). However, the efficiency of NMD varies; it is inefficient when a PTC is located downstream of the last exon junction complex (EJC). We used matched exome and transcriptome data from 9,769 human tumors to systematically elucidate the rules of NMD targeting in human cells. An integrated model incorporating multiple rules beyond the canonical EJC model explains approximately three-quarters of the non-random variance in NMD efficiency across thousands of PTCs. We also show that dosage compensation may mask the effects of NMD. Applying the NMD model identifies signatures of both positive and negative selection on NMD-triggering mutations in human tumors and provides a classification of tumor suppressor genes.
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            Human SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense-mediated mRNA decay.

            Nonsense-mediated mRNA decay (NMD) is a conserved surveillance mechanism that eliminates imperfect mRNAs that contain premature translation termination codons (PTCs) and code for nonfunctional or potentially harmful polypeptides. We show that a novel phosphatidylinositol 3-kinase-related protein kinase, hSMG-1, is a human ortholog of a product of Caenorhabditis elegans smg-1, one of seven smg genes involved in NMD. hSMG-1 phosphorylates hUPF1/SMG-2 in vivo and in vitro at specific serine residues in SQ motifs. hSMG-1 can associate with hUPF1/SMG-2 and other components of the surveillance complex. In particular, overexpression of a kinase-deficient point mutant of hSMG-1, hSMG-1-DA, results in a marked suppression of the PTC-dependent beta-globin mRNA degradation; whereas that of wild-type hSMG-1 enhances it. We also show that inhibitors of hSMG-1 induce the accumulation of truncated p53 proteins in human cancer cell lines with p53 PTC mutation. Taken together, we conclude that hSMG-1 plays a critical role in NMD through the direct phosphorylation of hUPF1/SMG-2 in the evolutionally conserved mRNA surveillance complex.
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              Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin.

              Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of full-length proteins. Previously we have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients, all carrying the W1282X nonsense mutation. Here we demonstrate that there are patients in whom the level of CF transmembrane conductance regulator (CFTR) nonsense transcripts is markedly reduced, while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. We further investigated the possibility that the nonsense-mediated mRNA decay (NMD) might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Our results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines derived from the same tissue. Variability was also found for 5 physiologic NMD substrates, RPL3, SC35 1.6 kb, SC35 1.7 kb, ASNS, and CARS. Importantly, our results demonstrate the existence of cells in which NMD of all transcripts was efficient and others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin. Together our results suggest that the efficiency of NMD might vary and hence have an important role in governing the response to treatments aiming to promote readthrough of PTCs in many genetic diseases.
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                Author and article information

                Journal
                Hum Mol Genet
                Hum. Mol. Genet
                hmg
                Human Molecular Genetics
                Oxford University Press
                0964-6906
                1460-2083
                01 June 2019
                23 January 2019
                23 January 2019
                : 28
                : 11
                : 1865-1871
                Affiliations
                [1 ]Development, Ageing and Disease, UCL Institute of Ophthalmology, London, UK
                [2 ]Department of Genetics, Moorfields Eye Hospital NHS Foundation Trust, London, UK
                [3 ]Department of Public Health & Primary Care, Norwich Medical School, University of East Anglia, Norwich, UK
                [4 ]Inserm U1051, Institute for Neurosciences of Montpellier, Montpellier, Montpellier Cedex, France
                [5 ]Department of Ophthalmology, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK
                Author notes
                To whom correspondence should be addressed at: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK. Email: m.moosajee@ 123456ucl.ac.uk
                Author information
                http://orcid.org/0000-0002-9626-8807
                Article
                ddz028
                10.1093/hmg/ddz028
                6522067
                30689859
                a9f33b84-5ffc-49b3-b58d-00144a5abd80
                © The Author(s) 2019. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 17 December 2018
                : 18 January 2019
                : 21 January 2019
                Page count
                Pages: 07
                Funding
                Funded by: Wellcome Trust 10.13039/100010269
                Funded by: Retina UK 10.13039/501100000310
                Funded by: Moorfields Eye Charity
                Funded by: National Eye Research Centre Charity
                Funded by: Fight for Sight UK 10.13039/501100000615
                Funded by: France Choroideremia
                Funded by: Choroideremia Research Foundation US
                Categories
                General Article

                Genetics
                Genetics

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