A Synonymous Single Nucleotide Polymorphism in ΔF508 CFTR Alters the Secondary Structure of the mRNA and the Expression of the Mutant Protein

Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (Delta F508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the Delta F508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the Delta F508 CFTR mRNA in the vicinity of the mutation. The consequence of Delta F508 CFTR mRNA "misfolding" is decreased translational rate. A synonymous single nucleotide variant of the Delta F508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native Delta F508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote Delta F508 CFTR misfolding. Therefore, we propose that mRNA "misfolding" contributes to Delta F508 CFTR protein misfolding and consequently to the severity of the human Delta F508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various Delta F508 homozygous CF patients.