Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator Combination
Genetic variants that introduce premature termination codons (PTCs) have remained difficult to therapeutically target due to lack of protein product. Nonsense mediated mRNA decay (NMD) targets PTC-bearing transcripts to reduce the potentially damaging effects of truncated proteins. Readthrough compo...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-09-01
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| Series: | Journal of Personalized Medicine |
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| Online Access: | https://www.mdpi.com/2075-4426/12/9/1448 |
| _version_ | 1797486128819863552 |
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| author | Alyssa Bowling Alice Eastman Christian Merlo Gabrielle Lin Natalie West Shivani Patel Garry Cutting Neeraj Sharma |
| author_facet | Alyssa Bowling Alice Eastman Christian Merlo Gabrielle Lin Natalie West Shivani Patel Garry Cutting Neeraj Sharma |
| author_sort | Alyssa Bowling |
| collection | DOAJ |
| description | Genetic variants that introduce premature termination codons (PTCs) have remained difficult to therapeutically target due to lack of protein product. Nonsense mediated mRNA decay (NMD) targets PTC-bearing transcripts to reduce the potentially damaging effects of truncated proteins. Readthrough compounds have been tested on PTC-generating variants in attempt to permit translation through a premature stop. However, readthrough compounds have not proved efficacious in a clinical setting due to lack of stable mRNA. Here, we investigate N-terminal variants in the cystic fibrosis transmembrane conductance regulator (<i>CFTR</i>) gene, which have been shown to escape NMD, potentially through a mechanism of alternative translation initiation at downstream AUG codons. We hypothesized that N-terminal variants in <i>CFTR</i> that evade NMD will produce stable transcript, allowing CFTR function to be restored by a combination of readthrough and protein modulator therapy. We investigate this using two cell line models expressing <i>CFTR</i>-expression minigenes (EMG; HEK293s and CFBEs) and primary human nasal epithelial (NE) cells, and we test readthrough compounds G418 and ELX-02 in combination with CFTR protein modulators. HEK293 cells expressing the variants E60X and L88X generate CFTR-specific core glycosylated products that are consistent with downstream translation initiation. Mutation of downstream methionines at codons 150 and 152 does not result in changes in CFTR protein processing in cells expressing L88X-<i>CFTR</i>-EMG. However, mutation of methionine at 265 results in loss of detectable CFTR protein in cells expressing E60X, L88X, and Y122X <i>CFTR</i>-EMGs, indicating that downstream translation initiation is occurring at the AUG codon at position M265. In HEK293 stable cells harboring L88X, treatment with readthrough compounds alone allows for formation of full-length, but misfolded CFTR protein. Upon addition of protein modulators in combination with readthrough, we observe formation of mature, complex-glycosylated CFTR. In CFBE and NE cells, addition of readthrough ELX-02 and modulator therapy results in substantial recovery of CFTR function. Our work indicates that N-terminal variants generate stable <i>CFTR</i> transcript due to translation initiation at a downstream AUG codon. Thus, individuals with CF bearing 5′ nonsense variants that evade NMD are ideal candidates for treatment with clinically safe readthrough compounds and modulator therapy. |
| first_indexed | 2024-03-09T23:28:48Z |
| format | Article |
| id | doaj.art-f2a9ca39c26946b793fdbc1c402cc9cf |
| institution | Directory Open Access Journal |
| issn | 2075-4426 |
| language | English |
| last_indexed | 2024-03-09T23:28:48Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Personalized Medicine |
| spelling | doaj.art-f2a9ca39c26946b793fdbc1c402cc9cf2023-11-23T17:13:18ZengMDPI AGJournal of Personalized Medicine2075-44262022-09-01129144810.3390/jpm12091448Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator CombinationAlyssa Bowling0Alice Eastman1Christian Merlo2Gabrielle Lin3Natalie West4Shivani Patel5Garry Cutting6Neeraj Sharma7McKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USAMcKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USADivision of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins Hospital, Baltimore, MD 21205, USAMcKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USADivision of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins Hospital, Baltimore, MD 21205, USADivision of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins Hospital, Baltimore, MD 21205, USAMcKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USAMcKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USAGenetic variants that introduce premature termination codons (PTCs) have remained difficult to therapeutically target due to lack of protein product. Nonsense mediated mRNA decay (NMD) targets PTC-bearing transcripts to reduce the potentially damaging effects of truncated proteins. Readthrough compounds have been tested on PTC-generating variants in attempt to permit translation through a premature stop. However, readthrough compounds have not proved efficacious in a clinical setting due to lack of stable mRNA. Here, we investigate N-terminal variants in the cystic fibrosis transmembrane conductance regulator (<i>CFTR</i>) gene, which have been shown to escape NMD, potentially through a mechanism of alternative translation initiation at downstream AUG codons. We hypothesized that N-terminal variants in <i>CFTR</i> that evade NMD will produce stable transcript, allowing CFTR function to be restored by a combination of readthrough and protein modulator therapy. We investigate this using two cell line models expressing <i>CFTR</i>-expression minigenes (EMG; HEK293s and CFBEs) and primary human nasal epithelial (NE) cells, and we test readthrough compounds G418 and ELX-02 in combination with CFTR protein modulators. HEK293 cells expressing the variants E60X and L88X generate CFTR-specific core glycosylated products that are consistent with downstream translation initiation. Mutation of downstream methionines at codons 150 and 152 does not result in changes in CFTR protein processing in cells expressing L88X-<i>CFTR</i>-EMG. However, mutation of methionine at 265 results in loss of detectable CFTR protein in cells expressing E60X, L88X, and Y122X <i>CFTR</i>-EMGs, indicating that downstream translation initiation is occurring at the AUG codon at position M265. In HEK293 stable cells harboring L88X, treatment with readthrough compounds alone allows for formation of full-length, but misfolded CFTR protein. Upon addition of protein modulators in combination with readthrough, we observe formation of mature, complex-glycosylated CFTR. In CFBE and NE cells, addition of readthrough ELX-02 and modulator therapy results in substantial recovery of CFTR function. Our work indicates that N-terminal variants generate stable <i>CFTR</i> transcript due to translation initiation at a downstream AUG codon. Thus, individuals with CF bearing 5′ nonsense variants that evade NMD are ideal candidates for treatment with clinically safe readthrough compounds and modulator therapy.https://www.mdpi.com/2075-4426/12/9/1448alternate startNMDreadthroughCFTR correctorsCFTR potentiatorstranscript stability |
| spellingShingle | Alyssa Bowling Alice Eastman Christian Merlo Gabrielle Lin Natalie West Shivani Patel Garry Cutting Neeraj Sharma Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator Combination Journal of Personalized Medicine alternate start NMD readthrough CFTR correctors CFTR potentiators transcript stability |
| title | Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator Combination |
| title_full | Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator Combination |
| title_fullStr | Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator Combination |
| title_full_unstemmed | Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator Combination |
| title_short | Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator Combination |
| title_sort | downstream alternate start site allows n terminal nonsense variants to escape nmd and results in functional recovery by readthrough and modulator combination |
| topic | alternate start NMD readthrough CFTR correctors CFTR potentiators transcript stability |
| url | https://www.mdpi.com/2075-4426/12/9/1448 |
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