U6 promoter‐enhanced GlnUAG suppressor tRNA has higher suppression efficacy and can be stably expressed in 293 cells

Koukuntla, Ramesh; Ramsey, W. Jay; Young, William A.; Link, Charles J.

doi:10.1002/jgm.2696

Cited by 8 publications

(6 citation statements)

References 47 publications

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“…This possibility is consistent with the viability of transgenic Drosophila and C. elegans organisms that over-express amber suppressor tRNA (Bianco, Townsley et al 2012, Chin 2014. Further, it has been shown that cell lines with stable expression of suppressor orthogonal tRNAs are viable (Koukuntla, Ramsey et al 2013). There are numerous examples of the use of codon-edited or suppressor tRNAs that when overexpressed in target cells or host-organisms result in biological activity that is not paired with toxicity, suggesting low suppression efficacy at bona fide termination codons, or that such events have limited cellular impact.…”

Section: Introductionsupporting

confidence: 70%

Engineered tRNA suppression of a CFTR nonsense mutation

et al. 2016

Preprint

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Ten percent of human diseases are caused by 'nonsense' mutations that lead to premature truncation of the protein reading frame. Small molecules that promote read-through of such PTC have significant clinical promise but current iterations suffer from low in vivo efficacy and the nonselective amino acid incorporation. Alternatively, while gene-modifying approaches, such as CRISPR/Cas9, represent a long-term solution, such treatments may be far from reaching the clinical setting. Building on previous work by our group and others, we describe a tRNA engineering approach that enables the conversion of an in frame nonsense stop mutation to the naturally occurring amino acid, thus rescuing the full-length wild type protein. Data is presented demonstrating the functionality of the approach with the rescue of CFTR W1282X, a human mutation that causes cystic fibrosis (CF). The stringency of the approach is confirmed by mass spectrometry in a model protein indicating the encoding of only tryptophan at the TGA suppression site. The data describe the first use of an edited tRNA to repair a CF causative mutation and serve a proof of principle for the eventual use of codon-edited tRNA for the therapeutic rescue of PTC disease codons.

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Section: Introductionsupporting

confidence: 70%

Engineered tRNA suppression of a CFTR nonsense mutation

et al. 2016

Preprint

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“…The fact is that the strength and functionality of tRNA promoters cannot be easily predicted bioinformatically. To overcome this problem, we took advantage of the fact that the endogenous U6 promoter was successfully used for in vivo expression of a suppressor tRNA in the past ( 56 ). Therefore, we designed our own U6 promoter with its natural PSE (proximal sequence element), TATA box and +1 position for production of all tRNAs of our interest.…”

Section: Resultsmentioning

confidence: 99%

“…Naturally, our newly developed tRNA overexpression system had to be validated for its functionality. Therefore, to demonstrate that an ectopically expressed tRNA is fully engaged with the process of translation, we exploited a specific form of human glutamine tRNA where the CUG* anticodon is mutated to CUA ( 56 ). This single point mutation renders this tRNA fully cognate for the UAG stop codon (Figure 2B , left column) and, therefore, it is expected to very efficiently suppress translation termination in vivo .…”

Section: Resultsmentioning

confidence: 99%

Increased expression of tryptophan and tyrosine tRNAs elevates stop codon readthrough of reporter systems in human cell lines

Beznosková

Bidou

Namy

et al. 2021

Nucleic Acids Research

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Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens.

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“…Ad 1) The U6 promoter seems most suitable. [12][13][14][15] Effective tRNA charging and recruitment are determined by aminoacyl tRNA synthetases (aaRS) and elongation factor eEF1α, which recognize specific tRNA identity elements but not always the stop codon, therefore special care must be taken to fine-tune the prospective therapeutic tRNAs. 16,17 Ad 2) Two recent studies have successfully pioneered a delivery route via a recombinant adeno-associated virus (rAAV).…”

Section: Could Specifically Modified Read-through-inducing Trnas Help?mentioning

confidence: 99%