The RNA-binding factor HuR is a ubiquitously expressed member of the Hu protein family that binds and stabilizes mRNAs containing AU-rich elements (AREs). Hu proteins share a common domain organization of two tandemly arrayed RNA recognition motifs (RRMs) near the N terminus, followed by a basic hinge domain and a third RRM near the C terminus. In this study, we engineered recombinant wild-type and mutant HuR proteins lacking affinity tags to characterize their ARE-binding properties. Using combinations of electrophoretic mobility shift and fluorescence anisotropy-based binding assays, we show that HuR can bind ARE substrates as small as 13 nucleotides with low nanomolar affinity, but forms cooperative oligomeric protein complexes on ARE substrates of at least 18 nucleotides in length. Analyses of deletion mutant proteins indicated that RRM3 does not contribute to high affinity recognition of ARE substrates, but is required for cooperative assembly of HuR oligomers on RNA. Finally, the hinge domain between RRM2 and RRM3 contributes significant binding energy to HuR⅐ARE complex formation in an ARE length-dependent manner. The hinge does not enhance RNA-binding activity by increased ion pair formation despite extensive positive charge within this region, and it does not thermodynamically stabilize protein folding. Together, the results define distinct roles for the HuR hinge and RRM3 domains in formation of cooperative HuR⅐ARE complexes in solution.

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AU-rich element RNA-binding protein 1 (AUF1) binding to AU-rich elements (AREs) in the 3-untranslated regions of mRNAs encoding many cytokines and other regulatory proteins modulates mRNA stability, thereby influencing protein expression. AUF1-mRNA association is a dynamic paradigm directed by various cellular signals, but many features of its function remain poorly described. There are four isoforms of AUF1 that result from alternative splicing of exons 2 and 7 from a common pre-mRNA. Preliminary evidence suggests that the different isoforms have varied functional characteristics, but no detailed quantitative analysis of the properties of each isoform has been reported despite their differential expression and regulation. Using purified recombinant forms of each AUF1 protein variant, we used chemical cross-linking and gel filtration chromatography to show that each exists as a dimer in solution. We then defined the association mechanisms of each AUF1 isoform for ARE-containing RNA substrates and quantified relevant binding affinities using electrophoretic mobility shift and fluorescence anisotropy assays. Although all AUF1 isoforms generated oligomeric complexes on ARE substrates by sequential dimer association, sequences encoded by exon 2 inhibited RNA-binding affinity. By contrast, the exon 7-encoded domain enhanced RNA-dependent protein oligomerization, even permitting cooperative RNA-binding activity in some contexts. Finally, fluorescence resonance energy transfer-based assays showed that the different AUF1 isoforms remodel bound RNA substrates into divergent structures as a function of protein:RNA stoichiometry. Together, these data describe isoform-specific characteristics among AUF1 ribonucleoprotein complexes, which likely constitute a mechanistic basis for differential functions and regulation among members of this protein family.

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 ...

Tristetraprolin (TTP) binds AU-rich elements (AREs) encoded within selected labile mRNAs and targets these transcripts for rapid cytoplasmic decay. RNA binding by TTP is mediated by an ϳ70-amino acid domain containing two tandemly arrayed CCCH zinc fingers. Here we show that a 73-amino acid peptide spanning the TTP zinc finger domain, denoted TTP73, forms a dynamic, equimolar RNA⅐peptide complex with a 13-nucleotide fragment of the ARE from tumor necrosis factor ␣ mRNA, which includes small but significant contributions from ionic interactions. Association of TTP73 with high affinity RNA substrates is accompanied by a large negative change in heat capacity without substantial modification of RNA structure, consistent with conformational changes in the peptide moiety during RNA binding. Analyses using mutant ARE substrates indicate that two adenylate residues located 3-6 bases apart within a uridylate-rich sequence are sufficient for high affinity recognition by TTP73 (K d <20 nM), with optimal affinity observed for RNA substrates containing AUUUA or AUUUUA. Linkage of conformational changes and binding affinity to the presence and spacing of these adenylate residues provides a thermodynamic basis for the RNA substrate specificity of TTP.Cytoplasmic mRNA stability is an important mechanism in the regulation of gene expression. In mammalian cells, some mRNAs encoding regulatory proteins like cytokines, inflammatory mediators, and oncoproteins decay very quickly, often exhibiting cytoplasmic half-lives of 30 min or less (reviewed in Refs. 1-3). Rapid turnover of these mRNAs is mediated by AU-rich elements (AREs), 1 a loosely defined collection of uridylate-rich sequences localized to the 3Ј-untranslated regions of many labile transcripts. The increased turnover rates of ARE-containing transcripts are typically manifested through acceleration of deadenylation, where the poly(A) tail is progressively shortened in a 3Ј 3 5Ј direction prior to extremely rapid degradation of the mRNA body (reviewed in Ref. 4).The mRNA-destabilizing activity of AREs is mediated by association of cytoplasmic trans-acting factors (reviewed in Ref. 5). Some factors, like members of the Hu family of RNA-binding proteins, prevent mRNA degradation (6, 7), whereas others, like AUF1 (8 -10) and KSRP (11), promote rapid decay of AREcontaining transcripts. Association of tristetraprolin (TTP) with ARE-containing mRNAs also promotes their rapid cytoplasmic catabolism, involving acceleration of deadenylation rates (12, 13). The significance of this mechanism is apparent from a TTP knock-out mouse model, where enhanced stability of tumor necrosis factor ␣ (TNF␣) mRNA in macrophages from TTP-deficient mice (14) produces constitutive enhancement of circulating TNF␣, ultimately leading to development of a systemic inflammatory syndrome (15).TTP is the prototype of the CCCH family of eukaryotic tandem zinc finger proteins (reviewed in Ref. 16). Interactions with selected AU-rich RNA substrates occur through the zinc finger domain, which is necessary and ...