The silent codon change I507‐ATC→ATT contributes to the severity of the ΔF508 CFTR channel dysfunction

Abstract

The most common disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the out-of-frame deletion of 3 nucleotides (CTT). This mutation leads to the loss of phenylalanine-508 (ΔF508) and a silent codon change (SCC) for isoleucine-507 (I507-ATC→ATT). ΔF508 CFTR is misfolded and degraded by endoplasmic reticulum-associated degradation (ERAD). We have demonstrated that the I507-ATC→ATT SCC alters ΔF508 CFTR mRNA structure and translation dynamics. By comparing the biochemical and functional properties of the I507-ATT and I507-ATC ΔF508 CFTR, we establish that the I507-ATC→ATT SCC contributes to the cotranslational misfolding, ERAD, and to the functional defects associated with ΔF508 CFTR. We demonstrate that the I507-ATC ΔF508 CFTR is less susceptible to the ER quality-control machinery during translation than the I507-ATT, although 27°C correction is necessary for sufficient cell-surface expression. Whole-cell patch-clamp recordings indicate sustained, thermally stable cAMP-activated Cl(-) transport through I507-ATC and unstable function of the I507-ATT ΔF508 CFTR. Single-channel recordings reveal improved gating properties of the I507-ATC compared to I507-ATT ΔF508 CFTR (NPo=0.45±0.037 vs. NPo=0.09±0.002; P<0.001). Our results signify the role of the I507-ATC→ATT SCC in the ΔF508 CFTR defects and support the importance of synonymous codon choices in determining the function of gene products.

Decoding mechanisms by which silent codon changes influence protein biogenesis and function

“…Such pauses were suggested to govern proper protein folding in vivo (Tinoco and Wen, 2009), and it was noted that mRNA structure also contributes to the length of translational pausing (Wen et al, 2008). Consistent with this finding, Bartoszewski and colleagues demonstrated that synonymous mutations altering mRNA structure also change translation dynamics, (Bartoszewski et al, 2010), leading to altered protein folding and function (Bartoszewski et al, 2010, Lazrak et al, 2013). …”

“…Analysis of the CFTR mRNA near the mutation revealed codon conservation across 43 species. The authors also analyzed three other synonymous mutations that were introduced to optimize plasmid propagation in the most widely used wild type and ΔF508 CFTR expression vectors (Rommens et al, 1991) and found no changes in predicted structure of the mRNAs (Lazrak et al, 2013). These results are consistent with the idea that not every synonymous codon change alters mRNA secondary structure.…”

Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD

“…These diseases include cystic fibrosis, amyotrophic lateral sclerosis (ALS), and Crohn's disease (Bartoszewski et al 2010;Lazrak et al 2013;Bali and Bebok 2015;Liu et al 2016). A single synonymous SNP in the form of a rare codon in the human multidrug resistance 1 (MDR1) gene was found to result in altered drug and inhibitor interactions (Kimchi-Sarfaty et al 2007).…”

Cystic fibrosis genetics: from molecular understanding to clinical application

“…Furthermore, the three-nucleotide deletion responsible for the F508del variant also causes a synonymous change in the triplet that encodes isoleucine at codon 507 (ATC-7ATT). The change alters the structure of the F508del-CFTR mRNA, which leads to a reduction in translation efficiency 19 . Thus, F508del could be assigned to at least three classes.…”