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Volume 271, Number 33, Issue of August 16, 1996 pp. 19871-19876
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Turnover and Translation of in Vitro Synthesized Messenger RNAs in Transfected, Normal Cells

(Received for publication, March 5, 1996, and in revised form, April 25, 1996)

Lakshman E. Rajagopalan and James S. Malter

From the  Department of Pathology and Laboratory Medicine and Comprehensive Cancer Center, University of Wisconsin, Madison, Wisconsin 53792-2472

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-3-UTR) was still more stable (t1/2 = 80 min) demonstrating the existence of non-AUUUA, 3-UTR destabilizing domains. Transfected -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.

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