title: UU/UA dinucleotide frequency reduction in coding regions results in increased mRNA stability and protein expression.
abstract: UU and UA dinucleotides are rare in mammalian genes and may offer natural selection against endoribonuclease-mediated mRNA decay. This study hypothesized that reducing UU and UA (UW) dinucleotides in the mRNA-coding sequence, including the codons and the dicodon boundaries, may promote resistance to mRNA decay, thereby increasing protein production. Indeed, protein expression from UW-reduced coding regions of enhanced green fluorescent protein (EGFP), luciferase, interferon-α, and hepatitis B surface antigen (HBsAg) was higher when compared to the wild-type protein expression. The steady-state level of UW-reduced EGFP mRNA was higher and the mRNA half-life was also longer. Ectopic expression of the endoribonuclease, RNase L, did not reduce the wild type or UW-reduced mRNA. A mutant form of the mRNA decay-promoting protein, tristetraprolin (TTP/ZFP36), which has a point mutation in the zinc-finger domain (C124R), was used. The wild-type EGFP mRNA but not the UW-reduced mRNA responded to the dominant negative action of the C124R ZFP36/TTP mutant. The results indicate the efficacy of the described rational approach to formulate a general scheme for boosting recombinant protein production in mammalian cells.
authors.affiliation: Department of Molecular Biomedicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
authors.forename: Maher
authors.initials: M
authors.lastname: Al-Saif
authors.forename: Khalid S A
authors.initials: KS
authors.lastname: Khabar
mesh_terms: D000818:Animals; D016466:CHO Cells; D003062:Codon / Q000235:genetics*; D006224:Cricetinae; D004722:Endoribonucleases / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006207:Half-Life; D006514:Hepatitis B Surface Antigens / Q000235:genetics; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics*; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D014162:Transfection; D051816:Tristetraprolin / Q000235:genetics / Q000378:metabolism
chemical_list: D003062:Codon; D006514:Hepatitis B Surface Antigens; D016898:Interferon-alpha; D009841:Oligonucleotides; D012333:RNA, Messenger; D051816:Tristetraprolin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease