UU/UA dinucleotide frequency reduction in coding regions results in increased mRNA stability and protein expression

Mol Ther. 2012 May;20(5):954-9. doi: 10.1038/mt.2012.29. Epub 2012 Mar 20.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Codon / genetics*
  • Cricetinae
  • Endoribonucleases / metabolism
  • Gene Expression*
  • Genetic Vectors
  • Green Fluorescent Proteins / genetics
  • HEK293 Cells
  • Half-Life
  • Hepatitis B Surface Antigens / genetics
  • Humans
  • Interferon-alpha / genetics
  • Luciferases / genetics
  • Mutation
  • Oligonucleotides / genetics*
  • Open Reading Frames / genetics*
  • RNA Stability / genetics*
  • RNA, Messenger / genetics*
  • Transfection
  • Tristetraprolin / genetics
  • Tristetraprolin / metabolism

Substances

  • Codon
  • Hepatitis B Surface Antigens
  • Interferon-alpha
  • Oligonucleotides
  • RNA, Messenger
  • Tristetraprolin
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Luciferases
  • Endoribonucleases
  • 2-5A-dependent ribonuclease