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 gene transfer into primary cultures of normal peripheral blood mononuclear cells. Transfected wild-type, human GM-CSF (hGM-AUUUA) mRNA decayed rapidly (t1/2 = 9 min), while a mutated version lacking AUUUA repeats (hGM-AUGUA) was significantly more stable (t1/2 = 30 min). A truncated GM-CSF mRNA lacking the entire 3'-UTR (hGM-Delta3'-UTR) was still more stable (t1/2 = 80 min) demonstrating the existence of non-AUUUA, 3'-UTR destabilizing domains. Transfected beta-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.
Rajagopalan
L E
LE
Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53792-2472, USA.
Malter
J S
JS
D001483:Base Sequence; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D005914:Globins / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic
D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D003609:Dactinomycin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D005914:Globins; D003513:Cycloheximide; D013755:Tetradecanoylphorbol Acetate