Identification of the amino acids inserted during suppression of CFTR nonsense mutations and determination of their functional consequences

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.

Positional effects of premature termination codons on the biochemical and biophysical properties of CFTR

“…During the read‐through, near‐cognate aminoacyl‐tRNAs are accommodated in the ribosome, leading to the insertion of a range of unintended amino acids into the nascent polypeptide chain; hence missense mutations at the site of the PTC are almost inevitable during the read‐through (Xue et al . ).…”

Structural mechanisms for defective CFTR gating caused by the Q1412X mutation, a severe Class VI pathogenic mutation in cystic fibrosis

“…; Xue et al . ), we suspect that this combination approach could be an effective treatment for patients carrying mutations other than Class II and III.…”

Rapid therapeutic advances in CFTR modulator science

“…Suppressors of PTCs are compounds which lead to the insertion of a near cognate aminoacyl tRNA in the A site of the eukaryotic ribosome, allowing translation to continue to the authentic termination codon and the production of a full‐length protein. These CFTR protein products following PTC suppression may or may not have full function based on the nature of the amino acid substitution coupled with the site of the substitution within the full‐length protein . Drugs that induce PTC read‐through and full length CFTR production have been tested in several contexts, including cell lines, primary airway cells from CF patients with PTCs, transgenic mice, and CF patients .…”

“…These CFTR protein products following PTC suppression may or may not have full function based on the nature of the amino acid substitution coupled with the site of the substitution within the full‐length protein . Drugs that induce PTC read‐through and full length CFTR production have been tested in several contexts, including cell lines, primary airway cells from CF patients with PTCs, transgenic mice, and CF patients . Some provocative small clinical trials have provided evidence that certain aminoglycosides, which are known to bind to the eukaryotic ribosome complex, can improve the function of CFTR in CF patients harboring PTC variants in CFTR .…”

“…Since this concept was first described in CF cells nearly two decades ago, accumulating data has helped the CF research community to better understand the many factors that contribute to PTC‐mediated CF . These include the importance of nonsense mediated decay impacting CFTR mRNA substrate levels, variable responsiveness of different PTCs to suppression, the role of the mRNA microenvironment (including nearby base pairs) in read‐through susceptibility, and the observation that some CFTR variants with PTCs late in the CFTR open reading frame retain partial function . This improved understanding of how cells regulate PTCs and their read‐through has led to the identification of new agents capable of producing more robust and predictable PTC suppression .…”