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 (DeltaF508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the DeltaF508 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 DeltaF508 CFTR mRNA in the vicinity of the mutation. The consequence of DeltaF508 CFTR mRNA "misfolding" is decreased translational rate. A synonymous single nucleotide variant of the DeltaF508 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 DeltaF508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote DeltaF508 CFTR misfolding. Therefore, we propose that mRNA "misfolding" contributes to DeltaF508 CFTR protein misfolding and consequently to the severity of the human DeltaF508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various DeltaF508 homozygous CF patients.
Highlights
Changes in mRNA secondary structures have major biological consequences [1,2,3,4]
Our results demonstrate that high fidelity computational methods [49, 50] and biochemical mRNA folding assays [27, 51] can provide background for studies investigating the dynamics of translation and protein folding
While several studies proposed that alterations in the secondary structures of mRNAs in the coding region may alter translation and protein folding [6, 7, 40, 41], there is limited experimental data in the literature to support this hypothesis
Summary
Molecular Modeling—mRNA structural models were created using the MFOLD algorithm [22, 28]. A Synonymous SNP Affects mRNA Structure and Protein Folding were used for in vitro transcription and translation experiments as described previously [29, 30]. Three individually synthesized and purified mRNA samples from each construct were tested. Following a 70% ethanol wash and air-drying, samples containing the RNA digests were solubilized in H2O and used in one-step semi-quantitative RTPCR (Applied Biosystems 4309169) to amplify WT and ⌬F508 CFTR-specific fragments. Primers were designed based on the differences in the predicted secondary structures of the WT and ⌬F508 CFTR mRNA fragments and the expected digests following RNase T1 digestion. Translation rates were determined from the reaction mixture based on the incorporation of [35S]methionine via TCA precipitation at different time points following addition of ATA.
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