UU/UA Dinucleotide Frequency Reduction in Coding Regions Results in Increased mRNA Stability and Protein Expression

• DOI: 10.1038/mt.2012.29
• Publication Date: 2012-03-20T09:58:15Z
• Authors: Maher Al-Saif; Khalid SA Khabar
• Keywords: 0301 basic medicine; RNA Stability; Genetic Vectors; Green Fluorescent Proteins; Oligonucleotides; Gene Expression; CHO Cells; Transfection; Open Reading Frames; 03 medical and health sciences; Cricetinae; Drug Discovery; Endoribonucleases; Genetics; Animals; Humans; RNA, Messenger; Codon; Luciferases; Molecular Biology; Pharmacology; Hepatitis B Surface Antigens; Interferon-alpha; HEK293 Cells; Mutation; Molecular Medicine; Original Article; Half-Life

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.