Synonymous codon usage affects the expression of wild type and F508del CFTR

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel composed of 1480 amino acids. The major mutation responsible for cystic fibrosis results in loss of amino acid residue, F508 (F508del). Loss of F508 in CFTR alters the folding pathway resulting in endoplasmic-reticulum-associated degradation. This study investigates the role of synonymous codon in the expression of CFTR and CFTR F508del in human HEK293 cells. DNA encoding the open reading frame (ORF) for CFTR containing synonymous codon replacements was expressed using a heterologous vector integrated into the genome. The results indicate that the codon usage greatly affects the expression of CFTR. While the promoter strength driving expression of the ORFs was largely unchanged and the mRNA half-lives were unchanged, the steady-state levels of the mRNA varied by as much as 30-fold. Experiments support that this apparent inconsistency is attributed to nonsense mediated decay independent of exon junction complex. The ratio of CFTR/mRNA indicates that mRNA containing native codons was more efficient in expressing mature CFTR as compared to mRNA containing synonymous high-expression codons. However, when F508del CFTR was expressed after codon optimization, a greater percentage of the protein escaped endoplasmic-reticulum-associated degradation resulting in considerable levels of mature F508del CFTR on the plasma membrane, which showed channel activity. These results indicate that codon usage has an effect on mRNA levels and protein expression, for CFTR, and likely on chaperone-assisted folding pathway, for F508del CFTR.

Keywords: CFTR; CFTR F508del; nonsense mediated decay; protein folding; synonymous codon usage.

Figure 1. CFTR Codon Usage and Molecular Model

The CAI index is plotted versus the residue number for codon optimized (hCAI) form of CFTR (A), the lCAI form (B), and the mCAI form (C). D. A model of CFTR showing the structural domains and the regions in mCAI where the clusters of codons with low CAI values are placed in the coding frame.

Figure 2. Dependence of expression of wild type CFTR and F508del CFTR on codon usage

A. Western blot analysis of total protein extracted from HEK293 cells using a monoclonal antibody directed against CFTR (593 from Univ. North Carolina). An antibody directed against the Na-K ATPase was used as an internal control. B. Quantification of the Western blot data. The data were quantified by measuring the intensity of the fluorescence of the secondary antibody using an Odyssey detector (Licor). Notice that the amount of total extracted protein varied per lane to allow analysis. Band B (blue) represents the core glycosylated form of CFTR, which is present in the ER. Band C (red) represents the complex glycosylated form of CFTR, which is present in the Golgi and plasma membrane. C. The expression of CFTR F508del was reduced by the addition of siRNA that targeted the 3′ nontranslated region of CFTR F508del mRNA. The left image is a Western blot analysis of the CFTR F508del from protein expressed in HEK293 cells in the presence and absence of 1nM siRNA. The siRNA reduced the expression of CFTR F508 del by about 45% but had no effect on the C:B ratio as shown in the bar graph (D.).

Figure 3. Effect of codon usage on steady state levels of mRNA

A. Fluorescence image of total cellular RNA after separation by agarose gel electrophoresis and stained with ethidium bromide. Only the region showing the 28S and 18S rRNA is shown. B. Northern blot analysis using a radiolabeled oligonucleotide probe directed against the 3′ non-translated region of the mRNAs. C. Quantitative analysis of the data from B. D/E. Relative ratio of Band C/mRNA derived from data in B. and C. Note the differences in the scales.

Figure 4. Analysis of the stability of the mRNA and relative promoter strength of the constructs

A. The cellular levels of the mRNA corresponding to CFTR and MAP3k14 were measured in HEK293 cells after addition of actinomycin D (2μM) and the results plotted in B. C. Nuclear run-on analysis to measure the relative promoter strength of the constructs using native codons, hCAI, mCAI, and lCAI. The amount of CFTR mRNA measured relative to the level of GAPDH mRNA, as described in Material and Methods.

Figure 5. CFTR mRNA is modulated by nonsense mediated decay

The cellular levels of mRNA were measured in HEK293 cells 48h after addition of siRNA against RENT1. The mRNA levels are relative to the levels of GAPDH mRNA and normalized relative to the levels of cellular levels of mRNA after addition a scrambled siRNA. The levels of MAP3k14 served as a control for a mRNA that is known to be under regulation by NMD.

Figure 6. Immunofluorescence Images of HEK293 Cells Expressing Wild Type and F508del CFTR

The images show the staining with wheat germ agglutinin conjugated with Rhodamine (red), (which stains the plasma membrane), mAb directed against CFTR followed by a secondary antibody labeled with Alexa-488 (green), and the merged images with co-localization in yellow.

Figure 7. Short circuit current (Isc) measured in native and hCAI F508del CFTR expressing FRT cells

A. Typical trace of current measurements for F508del CFTR. B. Current measurements for F508del CFTR expressed using native codons. C. Current measurements for F508del CFTR expressed using hCAI codons. Transfected cells were treated overnight with 10 μM C17 (a chemical corrector) or an equivalent amount of DMSO. For chemical correcting, C17 was added to the cells and the cells were incubated for 24 hrs before measuring conductance. Isc was measured before (baseline) and after the sequential addition of Forskolin (10 μM), PG-01 (10 μM) and Inh-172 (20 μM). Values are the means +/− SEM for n=6 filters. Note the axis scale.