Stabilization of granulocyte-macrophage colony-stimulating factor RNA in a human eosinophil-like cell line requires the AUUUA motifs.
Human eosinophils activated by calcium ionophore produce granulocyte-macrophage colony-stimulating factor (GM-CSF). In T lymphocytes GM-CSF messenger RNA (mRNA) stability is regulated by 3' untranslated region (UTR) adenosine-uridine-rich elements (AREs). We show endogenous GM-CSF mRNA is rapidly induced in an eosinophil cell-line (AML14.3D10) after activation with ionomycin. To calculate the decay rate of GM-CSF mRNA in activated cells, eosinophils were transfected with wild-type, full-length GM-CSF mRNA or a mutant version lacking the AUUUA motifs. In unstimulated cells, wild-type GM-CSF mRNA decayed with a half-life time (t1/2) of 6+/-2 min while the mutant decayed with a t1/2 of 20+/-4 min, demonstrating the dominant, destabilizing effect of multiple AUUUA motifs. Within 1 hr of activation by ionomycin, the half-life of transfected wild-type mRNA increased by 2.5-fold, which increased up to 4-fold after 2 hr of activation. The half-life of the mutant GM-CSF was unaffected by ionomycin, demonstrating that ionophore-mediated stabilization requires intact AUUUA motifs. Actinomycin D (ActD) stabilized wild-type GM-CSF mRNA as well, causing poly(A) tail elongation and translation inhibition. These data show that in eosinophil-like cell lines, GM-CSF mRNA is exquisitely unstable but can be markedly stabilized by calcium ionophore. Both effects require intact 3' UTR AREs.
Esnault
S
S
Department of Pathology and Laboratory Medicine, University of Wisconsin Hospital and Clinic, Madison 53792, USA.
Jarzembowski
J A
JA
Malter
J S
JS
D001665:Binding Sites; D002460:Cell Line; D003609:Dactinomycin / Q000494:pharmacology; D004804:Eosinophils / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D012333:RNA, Messenger
D012333:RNA, Messenger; D003609:Dactinomycin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor