INCREASED GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR MESSENGER-RNA INSTABILITY IN CORD VERSUS ADULT MONONUCLEAR-CELLS IS TRANSLATION-DEPENDENT AND ASSOCIATED WITH INCREASED LEVELS OF A-RICH ELEMENT-BINDING FACTOR(U)

The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA is fourfold lower in phorbol myristate acetate (PMA) + phytohema gglutinin (PHA)-activated mononuclear cells (MNC) from newborns compar ed with adults. The GM-CSF transcription rate is similar in umbilical cord and adult MNC, but transcript half-life is threefold lower in cor d activated MMC. Interaction of RNA binding proteins, such as the clon ed adenosine + uridine-rich element, binding factor, AUF1, with eight AUUUA motifs in the human GM-CSF mRNA 3'-untranslated region (GM-3'-UT R) has been implicated in regulating transcript stability. Translation al inhibition by cycloheximide (CHX) significantly increased GM-CSF mR NA accumulation and half-life by threefold in activated cord MNC, but had a minimal effect in activated adult MNC as compared with PMA + PHA alone. Electrophoretic mobility-shift assays with a P-32-labeled, 305 -nucleotide RNA comprising the GM-3'-UTR revealed two RNaseT1-resistan t, bound complexes that were almost twice as abundant in cord than in adult MNC extracts. Mobility-shift competition assays and RNaseT1 mapp ing localized the binding site of both complexes to a 52-nucleotide re gion containing seven of eight AUUUA motifs. Inclusion of AUF1 antiser um produced a supershifted complex at 35-fold higher levels in cord th an in adult MNC extracts, Extracts from the carcinoma cell line 5637, with extended GM-CSF mRNA half-life, also had very low levels of anti- AUF1 supershifted complex. Anti-AUF1 immunoblotting showed significant ly higher levels of two AUF1 protein isoforms and lower levels of one in cord than in adult MNC or 5637 extracts. These results suggest that destabilization of GM-CSF mRNA in cord MNC is translation-dependent a nd that increased levels of specific AUF1 isoforms in cord MNC may tar get transcripts for increased degradation, which could account in part for dysregulation of neonatal phagocytic immunity. (C) 1996 by The Am erican Society of Hematology.