In mammals, rapid mRNA turnover directed by AU-rich elements (AREs) is mediated by selective association of cellular ARE-binding proteins. These trans-acting factors display overlapping RNA substrate specificities and may act to either stabilize or destabilize targeted transcripts; however, the mechanistic features of AREs that promote preferential binding of one trans-factor over another are not well understood. Here, we describe a hairpin-like structure adopted by the ARE from tumor necrosis factor ␣ (TNF␣) mRNA that modulates its affinity for selected ARE-binding proteins. In particular, association of the mRNA-destabilizing factor p37 AUF1 was strongly inhibited by adoption of the higher order ARE structure, whereas binding of the inducible heat shock protein Hsp70 was less severely compromised. By contrast, association of the mRNA-stabilizing protein HuR was only minimally affected by changes in ARE folding. Consistent with the inverse relationship between p37 AUF1 binding affinity and the stability of ARE folding, mutations that stabilized the ARE hairpin also inhibited its ability to direct rapid mRNA turnover in transfected cells. Finally, phylogenetic analyses and structural modeling indicate that TNF␣ mRNA sequences flanking the ARE are highly conserved and may stabilize the hairpin fold in vivo. Taken together, these data suggest that local higher order structures involving AREs may function as potent regulators of mRNA turnover in mammalian cells by modulating trans-factor binding selectivity.The rate of mRNA turnover is highly variable among the cytoplasmic mRNA population and thus plays a significant role in regulating the steady-state concentrations of individual mRNA species available to program protein synthesis. In mammalian cells, different transcripts exhibit a range of decay kinetics spanning over 2 orders of magnitude, largely due to the presence of discrete cis-acting elements contained within each mRNA (1, 2). Most mRNAs encoding cytokines, inflammatory mediators, and proto-oncogenes are inherently unstable, often exhibiting cytoplasmic half-lives of 1 h or less. Rapid turnover of these transcripts is principally due to the activity of AU-rich elements (AREs), 1 a broad family of mRNA-destabilizing sequences localized to the 3Ј-untranslated regions (3Ј-UTRs) of many labile mRNAs (3). The intrinsic lability of ARE-containing mRNAs enables their cytoplasmic concentrations to be rapidly modulated following acute changes in their synthetic rates (4, 5). Additionally, modulation of ARE-directed mRNA decay pathways by selected signal transduction systems can regulate the cytoplasmic levels of some mRNAs independent of, or in concert with, changes in the synthetic rate (6 -8).The ability of AREs to direct mRNA turnover is mediated by the activity of selected ARE-binding proteins (9 -11). To date, over 25 such factors have been identified, although the regulatory significance of most remains unknown. Some proteins, including AUF1 (12-14), tristetraprolin (TTP) (15, 16), and KSRP (17, 18), appear ...