Translational readthrough of nonsense mutant TP53 by mRNA incorporation of 5-Fluorouridine

Palomar-Siles, Mireia; Heldin, Angelos; Zhang, Meiqiongzi; Strandgren, Charlotte; Yurevych, Viktor; Dinter, Jip T. van; Engels, Sem A.G.; Hofman, Damon A.; Öhlin, Susanne; Meineke, Birthe; Bykov, Vladimir J.N.; Heesch, Sebastiaan van; Wiman, Klas G.

doi:10.1038/s41419-022-05431-2

Cited by 15 publications

(21 citation statements)

References 57 publications

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“…The ochre suppression clonal cell lysate also featured a distinct band, suggesting a particularly strongly labeled endogenous protein. It is interesting to note, that endogenous opal codons do not appear to be more efficiently suppressed by tRNA Pyl than the other two stop codons, despite the opal codon being known to show highest leakiness, i.e., readthrough with endogenous tRNAs 33,39,40 . In summary, productive ochre and opal suppression is possible to achieve in stable settings.…”

Section: Resultsmentioning

confidence: 98%

“…It is interesting to note, that endogenous opal codons do not appear to be more efficiently suppressed by PylT than the other two stop codons, despite the opal codon being known to show highest leakiness, i.e. readthrough with endogenous tRNAs (Floquet et al 2012; Schmied et al 2014; Palomar-Siles et al 2022). In summary, productive ochre and opal suppression is possible to achieve in stable settings.…”

Section: Resultsmentioning

confidence: 99%

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Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Meineke

Caridha

Block

et al. 2023

Preprint

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Genetic code expansion via stop codon suppression is a powerful strategy to engineer proteins. Suppressor tRNAs are aminoacylated with noncanonical amino acids (ncAAs) by dedicated aminoacyl-tRNA synthetases (aaRS) and direct ncAA incorporation site-specifically during translation. These pairs of tRNA/aaRS must be orthogonal to the host's tRNAs, aaRS and natural amino acids. Pyrrolysyl-tRNA (PylT)/PylRS pairs from methanogenic archaea, as well as engineered tRNA/aaRS pairs derived from bacteria, are used for genetic code expansion in mammalian cells. Amber suppression is routinely achieved by transient introduction of the components leading to short-term and heterogeneous expression. Here, we demonstrate that stable integration of tRNA/aaRS genes allows for efficient, genetically encoded ncAA incorporation in diverse mammalian cell lines. We extend a general plasmid design and PiggyBac (PB) integration strategy developed for the Methanosarcina mazei PylT/PylRS pair to genomic integration of two tRNA/aaRS pairs of bacterial origin. We further explore suppression of ochre and opal stop codons and parallel incorporation of two distinct ncAAs, both accessible for click chemistry, by dual suppression in stable cell lines. Clonal selection allows for isolation of cells with high dual suppression efficiency and dual site-specific fluorescent labeling of a cell surface receptor using bioorthogonal click chemistries on live mammalian cells.

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Meineke

Caridha

Block

et al. 2023

Preprint

Self Cite

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“…Similarly, 5-Fluorouridine (FUr) was identified in a data mining effort of 47,000 anti-cancer drugs as potent readthrough-inducing compound [ 121 ]. Specifically, a search of the NCI-60 database for substances that suppress growth of a subset of tumor cells carrying p53 nonsense mutations (e.g., TP53 R213* UGA PTC) yielded 28 candidate compounds, among these the widely used anti-cancer drug 5-Fluorouracil (5-FU).…”

Section: Induction Of Nonsense Suppressionmentioning

confidence: 99%

“…Follow-up experiments revealed that the 5-FU metabolite 5-Fluorouridine (FUr) was the actual readthrough-inducing compound. FUr exerted its effect by direct incorporation into nonsense-bearing TP53 mRNA, where it facilitated the readthrough of the PTC but not of the canonical stop codon (as shown by Ribo-seq) [ 121 ]. Tests employing mutated superfolder GFP with a UGA, UAG or UAA PTC at position 150 further demonstrated that FUr induced readthrough of UGA or UAG but not UAA.…”

Section: Induction Of Nonsense Suppressionmentioning

confidence: 99%

Emerging Personalized Opportunities for Enhancing Translational Readthrough in Rare Genetic Diseases and Beyond

Wagner

Wießner

Friedrich

et al. 2023

IJMS

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Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa.

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“…This results in the production of abnormal RNAs (e.g., through altered splicing) 2 , aberrant post-transcriptional modifications (e.g. pseudo-uridylation) 7 , alteration of ribosome biogenesis or functions 8 , and aberrant translation-dependent events (e.g., stop-codon readthrough) 4,8 . However, the underlying mechanistic details of RNA-dependent 5FU cytotoxicity remains ill-defined 9 .…”

Section: Introductionmentioning

confidence: 99%

Ribosome Quality Control Mechanism Mitigates the Cytotoxic Impacts of Ribosome Collisions Induced by 5-Fluorouracil

Chatterjee,

Naeli,

Simms

et al. 2023

Preprint

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Translation of aberrant or damaged mRNAs results in ribosome stalling and collisions. The Ribosome Quality Control (RQC) mechanism detects collided ribosomes and removes aberrant mRNAs and nascent peptides, thus preventing their cytotoxic effects. Conversely, excessive or unresolved ribosome collisions can induce apoptosis. 5-Fluorouracil (5FU) forms the backbone of standard-of-care chemotherapeutic regimens for several types of cancer. Although best known for its incorporation into DNA and inhibition of thymidylate synthase, a major determinant of 5FU’s anticancer activity is its incorporation into RNAs. Nevertheless, the mechanism(s) underlying RNA-dependent 5FU cytotoxicity and the cellular response to its impact on RNA metabolism remain unclear. Here, we report a key role for RQC in mitigating the cytotoxic effects of 5FU-induced dysregulation of mRNA translation. We show that acute 5FU treatment results in the rapid induction of the mTOR signalling pathway, an enhanced rate of mRNA translation initiation, and increased ribosome collisions that trigger RQC. We also found that RQC deficiency, caused by the depletion of ZNF598, results in increased 5FU-induced cell death, a phenotype that is reversed by inhibition of mTOR or repression of mRNA translation initiation. Importantly, 5FU treatment enhances the expression of key RQC factors, including ZNF598 and GIGYF2, via an mTOR-dependent post-translational regulation mechanism. This acute adaptation likely mitigates the cytotoxic consequences of increased ribosome collisions upon 5FU treatment. Overall, our data indicate a heretofore unknown mTOR-dependent mechanism that augments the RQC process, mitigating the cytotoxicity of 5FU and undermining its anticancer efficacy.

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Translational readthrough of nonsense mutant TP53 by mRNA incorporation of 5-Fluorouridine

Cited by 15 publications

References 57 publications

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Emerging Personalized Opportunities for Enhancing Translational Readthrough in Rare Genetic Diseases and Beyond

Ribosome Quality Control Mechanism Mitigates the Cytotoxic Impacts of Ribosome Collisions Induced by 5-Fluorouracil

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