Modulation of granulocyte-macrophage colony-stimulating factor mRNA stability in vitro by the adenosine-uridine binding factor.
In vitro decay of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA was examined on polysomes prepared from normal human peripheral blood mononuclear cells stimulated with phorbol ester (TPA) and phytohemagglutinin for 14 h. GM-CSF mRNA decayed with a half-life of 90 min while 18 S rRNA was stable. RNA gel mobility assay performed on crude cytosolic lysate (S20) with radiolabeled AUUUA containing RNA identified a 42-kDa RNA-protein complex on SDS-polyacrylamide gel electrophoresis. The binding specificity was identical to that of the previously described adenosine-uridine binding factor (AUBF) (Malter, J. S. (1989) Science 246, 664-666). Further fractionation of the S20 cytosol through a sucrose gradient showed > 90% of AUBF activity associated with polysomes and < 10% with the S130 fraction. Solution phase RNAs containing AUUUA reiterations specifically competed for polysome-bound AUBF and accelerated the decay of GM-CSF mRNA (t1/2 = 17 min). We linked biotinylated AUUUA RNA to streptavidin magnetic beads and removed > 95% of polysome-associated AUBF. A decay system thus depleted of AUBF activity also showed accelerated decay of GM-CSF mRNA (t1/2 = 20 min). These data show that AUBF is preferentially located on polysomes and that its removal destabilizes GM-CSF mRNA. Therefore, AUBF likely prevents GM-CSF mRNA decay by binding to the AUUUA instability determinants in the 3'-untranslated region.
Rajagopalan
L E
LE
Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison 53792-2472.
Malter
J S
JS
D000241:Adenosine / Q000378:metabolism*; D001667:Binding, Competitive; D001710:Biotin / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008934:Mitogens / Q000494:pharmacology; D011132:Polyribosomes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D014529:Uridine / Q000378:metabolism*
D002352:Carrier Proteins; D008934:Mitogens; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C076141:adenosine-uridine binding factor; D001710:Biotin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000241:Adenosine; D014529:Uridine