Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells.
Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region.
Iwai
Y
Y
Division of Cytokine Biology, Food and Drug Administration, Bethesda, Maryland 20892.
Bickel
M
M
Pluznik
D H
DH
Cohen
R B
RB
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003208:Concanavalin A; D004273:DNA, Neoplasm / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D051379:Mice; D008934:Mitogens; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D012334:RNA, Neoplasm / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013945:Thymoma / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D004273:DNA, Neoplasm; D008934:Mitogens; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D003208:Concanavalin A; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate