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 (t( 1/2 ) = 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 (t( 1/2 ) = 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.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - Sep 30 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology