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Updated: Mar 1, 2026
Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein
Published on: March 9, 2015
Identification of the amino acids inserted during suppression of CFTR nonsense mutations and determination of their
Xiaojiao Xue1,2, Venkateshwar Mutyam2,3, Amita Thakerar4
1Department of Biochemistry and Molecular Genetics.
Abstract:
In-frame premature termination codons (PTCs) account for ∼11% of all disease-associated mutations. PTC suppression therapy utilizes small molecules that suppress translation termination at a PTC to restore synthesis of a full-length protein. PTC suppression is mediated by the base pairing of a near-cognate aminoacyl-tRNA with a PTC and subsequently, the amino acid becomes incorporated into the nascent polypeptide at the site of the PTC. However, little is known about the identity of the amino acid(s) inserted at a PTC during this process in mammalian cells, or how the surrounding sequence context influences amino acid incorporation. Here, we determined the amino acids inserted at the cystic fibrosis transmembrane conductance regulator (CFTR) W1282X PTC (a UGA codon) in the context of its three upstream and downstream CFTR codons during G418-mediated suppression. We found that leucine, cysteine and tryptophan are inserted during W1282X suppression. Interestingly, these amino acids (and their proportions) are significantly different from those recently identified following G418-mediated suppression of the CFTR G542X UGA mutation. These results demonstrate for the first time that local mRNA sequence context plays a key role in near-cognate aminoacyl-tRNA selection during PTC suppression. We also found that some variant CFTR proteins generated by PTC suppression exhibit reduced maturation and activity, indicating the complexity of nonsense suppression therapy. However, both a CFTR corrector and potentiator enhanced activity of protein variants generated by G418-mediated suppression. These results suggest that PTC suppression in combination with CFTR modulators may be beneficial for the treatment of CF patients with PTCs.
Insights
Premature termination codons (PTCs) cause disease. PTC suppression therapy inserts amino acids at PTCs, but context matters. This study shows sequence context influences amino acid insertion during cystic fibrosis transmembrane conductance regulator (CFTR) W1282X PTC suppression.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Premature termination codons (PTCs) represent a significant class of disease-causing mutations.
- Nonsense suppression therapy aims to restore protein synthesis by suppressing translation termination at PTCs.
- The identity of amino acids incorporated during PTC suppression and the influence of mRNA sequence context remain poorly understood in mammalian cells.
Purpose of the Study:
- To investigate the amino acids inserted at the cystic fibrosis transmembrane conductance regulator (CFTR) W1282X PTC during G418-mediated suppression.
- To determine the impact of local mRNA sequence context on amino acid incorporation at the W1282X PTC.
- To assess the functional consequences of PTC suppression and the potential of combination therapy with CFTR modulators.
Main Methods:
- Site-directed mutagenesis to create the CFTR W1282X PTC.
- G418-mediated nonsense suppression in mammalian cells.
- Analysis of amino acid incorporation at the PTC.
- Assessment of CFTR protein maturation and activity.
Main Results:
- Leucine, cysteine, and tryptophan were identified as the primary amino acids inserted during W1282X PTC suppression.
- The proportions of inserted amino acids differed significantly from those observed in other CFTR PTCs, highlighting the role of sequence context.
- Some PTC-suppressed CFTR variants showed reduced maturation and activity, but their function was enhanced by CFTR correctors and potentiators.
Conclusions:
- Local mRNA sequence context critically influences near-cognate aminoacyl-tRNA selection during PTC suppression in mammalian cells.
- PTC suppression therapy can generate functional CFTR variants, but protein maturation and activity are complex.
- Combining PTC suppression with CFTR modulators may offer a promising therapeutic strategy for cystic fibrosis patients with PTCs.
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