title: 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.
authors: Department of Biochemistry and Molecular Biology, Rosalind Franklin University, The Chicago Medical School, North Chicago, IL 60064, USA; Department of Physiology and Biophysics, Rosalind Franklin University, The Chicago Medical School, North Chicago, IL 60064, USA.
authors: Kalpit
authors: K
authors: Shah
authors: Department of Physiology and Biophysics, Rosalind Franklin University, The Chicago Medical School, North Chicago, IL 60064, USA.
authors: Yi
authors: Y
authors: Cheng
authors: Department of Biochemistry and Molecular Biology, Rosalind Franklin University, The Chicago Medical School, North Chicago, IL 60064, USA.
authors: Brian
authors: B
authors: Hahn
authors: Department of Physiology and Biophysics, Rosalind Franklin University, The Chicago Medical School, North Chicago, IL 60064, USA.
authors: Robert
authors: R
authors: Bridges
authors: Department of Physiology and Biophysics, Rosalind Franklin University, The Chicago Medical School, North Chicago, IL 60064, USA.
authors: Neil A
authors: NA
authors: Bradbury
authors: Department of Biochemistry and Molecular Biology, Rosalind Franklin University, The Chicago Medical School, North Chicago, IL 60064, USA. Electronic address: david.mueller@rosalindfranklin.edu.
authors: David M
authors: DM
authors: Mueller
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D003062:Codon / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics*; D017510:Protein Folding; D021381:Protein Transport; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion
chemical_list: C505032:CFTR protein, human; D002712:Chlorides; D003062:Codon; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: CFTR; CFTR F508del; nonsense mediated decay; protein folding; synonymous codon usage