Tumor necrosis factor-alpha (TNF-alpha) is a potent cytokine that is central to normal immune responses as well as autoimmune inflammatory diseases. The production of TNF-alpha protein is thus tightly regulated at multiple levels. Translational control is one of the means by which TNF-alpha. production is repressed in unstimulated cells. To examine the mechanism by which the translation of TNF-alpha mRNA transcripts is repressed, we have used an in vitro translation system. The AU-rich element (ARE) in the 3' UTR of TNF-alpha transcripts was sufficient to confer translational repression. This effect was observed using transcripts containing a 5' in 7 G cap but not uncapped transcripts, and was independent of a poly(A) tail. Sucrose gradient analysis revealed that ARE-containing transcripts were present at relatively lower amounts in SOS-associated fractions and higher amounts in non-ribosome-bound RNA fractions, with no accumulation of 48S-associated transcripts. ARE-mediated translational repression was competitively inhibited by ARE-containing transcripts. These data indicate that a TNF-alpha ARE-binding trans-acting factor(s) inhibits the association of the 43S complex with RNA transcripts. (c) 2005 Elsevier Inc. All rights reserved.