Turnover and translation of in vitro synthesized messenger RNAs in transfected, normal cells

We have developed a novel system to examine intracellular mRNA decay pathways in the absence of transcriptional blockade. In vitro transcribed, capped, and adenylated granulocyte-macrophage colony stimulating factor (GM·CSF) or globin mRNAs were introduced by particle-mediated gent transfer into primary cultures of normal peripheral blood mononuclear cells. Transfected wild-type, human GM-CSF (hGM-AUUUA) mRNA decayed rapidly (t( 1/4 ) = 9 min), while a mutated version lacking AUUUA repeats (hGM-AUGUA) was significantly more stable (t( 1/4 ) = 30 min). A truncated GM-CSF mRNA lacking the entire 3'-UTR (hGM- Δ3'-UTR) was still more stable (t( 1/4 ) = 80 min) demonstrating the existence of non-AUUUA, 3'-UTR destabilizing domains. Transfected β-globin mRNA was very stable, decaying with a half-life of >360 min. Transfected mRNAs were >90% polysome associated with transgenic protein detectable within 15 min of transfection. The most stable GM-CSF mRNAs were not associated with maximal GM- CSF protein production. Agents known or hypothesized to interfere with mRNA decay, including cycloheximide, phorbol ester, or actinomycin D, stabilized both hGM-AUUUA and hGM-AUGUA mRNAs. These data demonstrate the presence of 3'-UTR, destabilizing, and translational regulatory elements outside of the AUUUA repeats and unambiguously show that actinomycin D at concentrations commonly used to inhibit transcription stabilizes cytokine mRNAs.