A synonymous single nucleotide polymorphism in DeltaF508 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 (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.

FIGURE 1.

Alterations in ΔF508 CFTR mRNA secondary structure as shown by theoretical modeling and circular dichoism spectroscopy. CFTR gene sequences for WT (A), ΔF508 (Ile507ATT) (B), and ΔF508 (Ile507ATC) (C) and corresponding amino acid sequences are shown (top panels). Theoretical models of the mRNA secondary structures in this region (bottom panel) are shown. The synonymous SNP (T) introduced by the mutation (*) and the exchanged nucleotide in the variant (Ile507ATC) ΔF508 CFTR are illustrated in the top panel. Nucleotides 1503 and 1540 (WT), 1498, 1528, and 1561 (ΔF508 CFTR Ile508ATT), and 1503 and 1537 (ΔF508 CFTR Ile507ATC) are labeled for easy comparison of the models (A in the ATG start codon = 1; the CTT deletion corresponds to nucleotides 1521–23); Ile-507 is labeled by brackets; ***: the ΔF508 mutation site. Full-length mRNA secondary structures (MFOLD) are shown in supplemental Fig. S1, A–C). D, CD spectra and differential CD spectrum of the WT and ΔF508 (Ile507ATT) CFTR mRNAs. The results are expressed in molar ellipticity [θ] as a function of the wavelength (λ); representative spectra of three individual experiments. Spectra were obtained from six individual runs of the same mRNA preparation. Maximum and minimum values and shifts in the wavelengths (± S.D. (n = 18)) are indicated by black and gray arrows, respectively.

FIGURE 2.

Theoretical models of CFTR mRNA fragments and RNA folding assay results. A, WT CFTR fragment model. B, ΔF508 (Ile507ATT) CFTR fragment model. C, variant (Ile507ATC) ΔF508 CFTR fragment model. Ile-507 (red); the CTT deletion site (CUU) is indicated by brackets; the structures that are identical to the full-length mRNA models are highlighted in gray. Nucleotides corresponding to the full-length sequences are labeled green; blue lines represent the projected PCR products predicted following T1 RNase digestion; single-stranded Gs (T1 site) are indicated by X; primers 1 and 2: complementary RT-PCR primer sequences used to amplify WT or ΔF508 (Ile507ATT) CFTR-specific products. D, RT-PCR amplification of WT and ΔF508 (Ile507ATT) CFTR-specific mRNA fragments following RNase T1 digestion. Arrows indicate WT (61 bp) ΔF508 (55 bp) fragment structure specific PCR products in samples digested with 0.05 units of RNase T1. M: molecular weight marker. A representative gel of four individual experiments is shown.

FIGURE 3.

CFTR mRNA half-life and translational rate measurements. A, WT and ΔF508 CFTR mRNA half-life measurements. Cells expressing HeLaWT and HeLaΔF cells were treated with actinomycin D to inhibit transcription. RNA was isolated at the time points specified and relative CFTR mRNA levels were measured by real time RT-PCR. CFTR mRNA levels at the start of the experiment (T₀) were set as 100%. Results are plotted as % of CFTR mRNA/t₀. CFTR mRNA half-lives were calculated from the exponential decay based on trend line equation C/C0 = e^−kdt (C and C₀ are mRNA amounts after time t and t₀, respectively, and k_d is the mRNA decay constant; R² = 0.99). Results were validated in three independent experiments. B, in vitro CFTR translation rates in the presence of microsomes and ATA. ATA was added 10 min after initiation of translation. Incorporation of [³⁵S]methionine was measured 15, 20, 25, 30, 50, and 60 min following addition of ATA. *, significant differences were found in ³⁵S incorporation levels between WT and ΔF508 (Ile507ATT) CFTR translation at 15, 20, 25, and 30 min; n = 6; p < 0.01. No significant differences were found in the translation rates of WT and variant ΔF508 (Ile507ATC) CFTR.

FIGURE 4.

Expression of native (Ile507ATT) and variant (Ile507ATC) ΔF508 CFTR in 293F cells. A, CFTR expression levels at 37 and 27 °C. 293F cells expressing native or variant ΔF508 CFTR were induced with 4 ng/ml doxacyclin and cultured at 37 or 27 °C for 4 h. Cells were lysed in RIPA, and proteins were separated by PAGE (6%). CFTR was detected with MM13–4 monoclonal antibody and ECL. Higher expression of Band B CFTR in the Ile507ATC ΔF508 CFTR-expressing cells at 37 °C and significant levels of Band C CFTR following a 4-h culture at 27 °C are shown. B, native (Ile507ATT) ΔF508 CFTR Band B levels increase at 27 °C. Changes in Band B levels of native (Ile507ATT) and variant (Ile507ATC) ΔF508 CFTR were measured following a 4 h culture at 27 °C by Western blotting and densitometry. Results are expressed as fold increase in Band B density at 27 °C compared with 37 °C controls; n = 3. Inlay, representative gels; black arrowheads: Band B CFTR. C, CFTR mRNA levels following doxacyclin induction and 4 h at 27 °C. Ile507ATT and Ile507ATC ΔF508 CFTR mRNA levels were compared in real time RT-PCR. mRNA levels in the variant (Ile507ATC) cells are plotted as fold increase over native (Ile507ATT) ΔF508 CFTR, (n = 6). D, CFTR protein levels in serial dilutions of variant, Ile507ATC ΔF508 CFTR expressing cell lysates. Cellular proteins were isolated following doxacyclin induction and 4-h culture at 27 °C. Western blots were performed as described in A. Enhanced Band B and significant Band C formation in the Ile507ATC samples compared with native (Ile507ATT) ΔF508 CFTR.