Abstract
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.
Highlights
Regulated mRNA stability appears to depend on the interaction between intrinsic, cis-acting elements and trans-acting factors
We examined if particle-mediated gene transfer (PMGT) could be used to deliver in vitro transcribed mRNAs directly into cells, whereupon their decay kinetics would be assessed without the addition of actinomycin D (Act D) or DRB
The increased stability of hGM⌬3Ј-UTR compared with hGM-AUGUA mRNA suggests the existence of additional instability determinants in the 3Ј-untranslated region of granulocyte-macrophage colony-stimulating factor (GM-CSF)
Summary
The costs of publication of this article were defrayed in part by mRNA decay rates in the absence of transcriptional blockade. We and others have shown that particle-mediated gene transfer (PMGT) is an effective and versatile method for transfecting a range of normal cells and transformed cell lines irrespective of cell cycle position [10, 33]. While the efficiency is modest (5–10%) for resting, normal T lymphocytes [33], successfully transfected cells generate ample transgenic mRNA and protein. We investigated if normal resting and transformed cells could be transfected with in vitro synthesized mRNAs using particle-mediated gene transfer. Relative decay rates were preserved between unstable and stable mRNAs and compounds with established, but mechanistically unknown effects on mRNA decay such as cycloheximide, phorbol ester (TPA), or Act D had the anticipated effects on transgenic mRNAs
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