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

Abstract

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 (⌬F508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ⌬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 ⌬F508 CFTR mRNA in the vicinity of the mutation. The consequence of ⌬F508 CFTR mRNA "misfolding" is decreased translational rate. A synonymous single nucleotide variant of the ⌬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 ⌬F508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ⌬F508 CFTR misfolding. Therefore, we propose that mRNA "misfolding" contributes to ⌬F508 CFTR protein misfolding and consequently to the severity of the human ⌬F508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ⌬F508 homozygous CF patients.

Functions of transmembrane domain 3 of human melanocortin-4 receptor

“…Decreased total receptor expression has been reported previously for some missense GPCR mutations including the MC4R (Fan & Tao 2009, Wang & Tao 2011, Yang & Tao 2012, Zhang et al 2012, even for some synonymous mutations (Duan et al 2003, Bartoszewski et al 2010. We therefore further studied whether the decreased total receptor expression was due to accelerated protein degradation (Yang et al 2011).…”

“…These results suggested that increased degradation by the proteasome or lysosome was not responsible for the diminished expression of these mutants. Decreased protein synthesis due to altered secondary structure of mRNA (Duan et al 2003, Bartoszewski et al 2010) of those MC4R mutants might be responsible for the decreased receptor expression, although posttranscriptional mechanisms might also be involved. Further studies are needed to address the detailed causes of the observed decreased total MC4R expression.…”

Kinetic modelling indicates that fast-translating codons can coordinate cotranslational protein folding by avoiding misfolded intermediates

“…Understanding the process by which proteins fold during their biosynthesis is one of the most fundamental problems in molecular biology, as it is crucial to enable their biological function 3,40 , and its failure can result in their misfolding [40][41][42] , malfunction 7 and aggregation 3,4,43 , events that are associated with a wide range of severe health conditions including neurodegenerative disease 44 . A key challenge in this context is to interpret and predict the influence of individual codon translation rates on cotranslational protein folding and misfolding.…”

Improving Cell Surface Functional Expression of F508 CFTR: A Quest for Therapeutic Targets

“…A recent study revealed that the synonymous codon change of I507 in the F508 allele can cause mRNA misfolding, leading to reduced rate of translation and/or impaired cotranslational folding of F508 CFTR (Bartoszewski et al, 2010). Therefore, codon-dependent mRNA folding represents a new mechanism by which F508 CFTR expression can be regulated.…”