MAPPING OF A MINIMAL AU-RICH SEQUENCE REQUIRED FOR LIPOPOLYSACCHARIDE-INDUCED BINDING OF A 55-KDA PROTEIN ON TUMOR-NECROSIS-FACTOR-ALPHA MESSENGER-RNA

In monocyte/macrophage cells, the translation of tumor necrosis factor -alpha (TNF-alpha) mRNA is tightly controlled. In unstimulated cells, TNF-alpha mRNA is translationally repressed. However, upon stimulation of the cells with various agents (e.g. lipopolysaccharides (LPS) and viruses), this repression is overcome and translation occurs. The key element in this regulation is the AU-rich sequence present in the 3'-u ntranslated region of TNF-alpha mRNA, Several groups have described th e binding of proteins on AU-rich elements (AREs), We have previously r eported the binding of two cytosolic protein complexes (1 and 2) to th e TNF-alpha mRNA ARE, one of which (complex 2) is observed only follow ing induction of TNF-alpha production by LPS, In this report, we have demonstrated that complex 1 involves a long fragment of the ARE, where as the formation of the LPS-inducible complex 2 requires a minimal seq uence which corresponds to the nonanucleotide UUAUUUAUU. Furthermore, we show that the RNA-binding protein involved in complex 2 has an appa rent molecular mass of 55 kDa, Finally, we tested other AREs for their ability to form com plex 2. We observed that the ARE derived from gra nulocyte/macrophage colony-stimulating factor mRNA which does contain the nonanucleotide, is able to sustain the LPS-induced binding of the 55-kDa protein. However, c-myc mRNA which does not contain the nonanuc leotide, is unable to promote the formation of any LPS-induced complex .