Selective degradation of AU-rich mRNAs promoted by the p37 AUF1 protein isoform

An AU-rich element (ARE) consisting of repeated canonical AUUUA motifs confers rapid degradation to many cytokine mRNAs when present in the 3' untranslated region. Destabilization of mRNAs with AREs (ARE-mRNAs) is consistent with the interaction of ARE-binding proteins such as tristetraprolin and the four AUF1 isoforms. However, the association of the AUF1-mRNA interaction with decreased ARE-mRNA stability is correlative and has not been directly tested. We therefore determined whether overexpression of AUF1 isoforms promotes ARE-mRNA destabilization and whether AUF1 isoforms are limiting components for ARE-mRNA decay. We show that the p37 AUF1 isoform and, to a lesser extent, the p40 isoform possess ARE-mRNA-destabilizing activity when overexpressed. Surprisingly, overexpressed p37 AUF1 also destabilized reporter mRNAs containing a noncanonical but AU-rich 3' untranslated region. Since overexpressed p37 AUF1 could interact in vivo with the AU-rich reporter mRNA, AUF1 may be involved in rapid turnover of mRNAs that lack canonical AREs. Moreover, overexpression of p37 AUF1 restored the ability of cells to rapidly degrade ARE-mRNAs when that ability was saturated and inhibited by overexpression of ARE-mRNAs. Finally, activation of ARE-mRNA decay often involves a translation-dependent step, which was eliminated by overexpression of p37 AUF1. These data indicate that the p37 AUF1 isoform and, to some extent, the p40 isoform are limiting factors that facilitate rapid decay of AU-rich mRNAs.     

Regulation of A + U-rich element-directed mRNA turnover involving reversible phosphorylation of AUF1

Proteins binding A + U-rich elements (AREs) contribute to the rapid cytoplasmic turnover of mRNAs containing these sequences. However, this process is a regulated event and may be accelerated or inhibited by myriad signal transduction systems. For example, monocyte adherence at sites of inflammation or tissue injury is associated with inhibition of ARE-directed mRNA decay, which contributes to rapid increases in cytokine and inflammatory mediator production. Here, we show that acute exposure of THP-1 monocytic leukemia cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate mimics several features of monocyte adherence, including rapid induction and stabilization of ARE-containing mRNAs encoding interleukin-1 beta and tumor necrosis factor alpha. Additionally, TPA treatment alters the activity of cytoplasmic complexes that bind AREs, including complexes containing the ARE-specific, mRNA-destabilizing factor, AUF1. Analyses of AUF1 from control and TPA-treated cells indicated that post-translational modifications of the major cytoplasmic isoform, p40AUF1, are altered concomitant with changes in RNA binding activity and stabilization of ARE-containing mRNAs. In particular, p40AUF1 recovered from polysomes was phosphorylated on Ser83 and Ser87 in untreated cells but lost these modifications following TPA treatment. We propose that selected signal transduction pathways may regulate ARE-directed mRNA turnover by reversible phosphorylation of polysome-associated p40AUF1.     

Phosphorylation of p40AUF1 regulates binding to A + U-rich mRNA-destabilizing elements and protein-induced changes in ribonucleoprotein structure

Messenger RNA turnover directed by A + U-rich elements (AREs) involves selected ARE-binding proteins. Whereas several signaling systems may modulate ARE-directed mRNA decay and/or post-translationally modify specific trans-acting factors, it is unclear how these mechanisms are linked. In THP-1 monocytic leukemia cells, phorbol ester-induced stabilization of some mRNAs containing AREs was accompanied by dephosphorylation of Ser83 and Ser87 of polysome-associated p40AUF1. Here, we report that phosphorylation of p40AUF1 influences its ARE-binding affinity as well as the RNA conformational dynamics and global structure of the p40AUF1-ARE ribonucleoprotein complex. Most notably, association of unphosphorylated p40AUF1 induces a condensed RNA conformation upon ARE substrates. By contrast, phosphorylation of p40AUF1 at Ser83 and Ser87 inhibits this RNA structural transition. These data indicate that selective AUF1 phosphorylation may regulate ARE-directed mRNA turnover by remodeling local RNA structures, thus potentially altering the presentation of RNA and/or protein determinants involved in subsequent trans-factor recruitment.     

Assembly of AUF1 oligomers on U-rich RNA targets by sequential dimer association

Many labile mammalian mRNAs are targeted for rapid cytoplasmic turnover by the presence of A + U-rich elements (AREs) within their 3'-untranslated regions. These elements are selectively recognized by AUF1, a component of a multisubunit complex that may participate in the initiation of mRNA decay. In this study, we have investigated the recognition of AREs by AUF1 in vitro using oligoribonucleotide substrates. Gel mobility shift assays demonstrated that U-rich RNA targets were specifically bound by AUF1, generating two distinct RNA-protein complexes in a concentration-dependent manner. Chemical cross-linking revealed the interaction of AUF1 dimers to form tetrameric structures involving protein-protein interactions in the presence of high affinity RNA targets. From these data, a model of AUF1 association with AREs involving sequential dimer binding was developed. Using fluorescent RNA substrates, binding parameters of AUF1 dimer-ARE and tetramer-ARE equilibria were evaluated in solution by fluorescence anisotropy measurements. Using two AUF1 deletion mutants, sequences C-terminal to the RNA recognition motifs are shown to contribute to the formation of the AUF1 tetramer.ARE complex but are not obligate for RNA binding activity. Kinetic studies demonstrated rapid turnover of AUF1.ARE complexes in solution, suggesting that these interactions are very dynamic in character. Taken together, these data support a model where ARE-dependent oligomerization of AUF1 may function to nucleate the formation of a trans-acting, RNA-destabilizing complex in vivo.     

Folding of A+U-rich RNA elements modulates AUF1 binding. Potential roles in regulation of mRNA turnover

In mammals, A+U-rich elements (AREs) are potent cis-acting determinants of rapid cytoplasmic mRNA turnover. Recognition of these sequences by AUF1 is associated with acceleration of mRNA decay, likely involving recruitment or assembly of multi-subunit trans-acting complexes. Previously, we demonstrated that AUF1 deletion mutants formed tetramers on U-rich RNA substrates by sequential addition of protein dimers (Wilson, G. M., Sun, Y., Lu, H., and Brewer, G. (1999) J. Biol. Chem. 274, 33374-33381). Here, we show that binding of the full-length p37 isoform of AUF1 to these RNAs proceeds via a similar mechanism, allowing delineation of equilibrium binding constants for both stages of tetramer assembly. However, association of AUF1 with the ARE from tumor necrosis factor (TNFalpha) mRNA was significantly inhibited by magnesium ions. Further fluorescence and hydrodynamic experiments indicated that Mg(2+) induced or stabilized a conformational change in the TNFalpha ARE. Based on the solution of parameters describing both the protein-RNA and Mg(2+)-RNA equilibria, we present a dynamic, global equilibrium binding model describing the relationship between Mg(2+) and AUF1 binding to the TNFalpha ARE. These studies provide the first evidence that some AREs may adopt higher order RNA structures that regulate their interaction with trans-acting factors and indicate that mRNA structural remodeling has the potential to modulate the turnover rates of some ARE-containing mRNAs.     

Tissue distribution of AU-rich mRNA-binding proteins involved in regulation of mRNA decay

Short lived cytokine and proto-oncogene mRNAs are destabilized by an A+U-rich element (ARE) in the 3'-untranslated region. Several regulatory proteins bind to AREs in cytokine and proto-oncogene mRNAs, participate in inhibiting or promoting their rapid degradation of ARE mRNAs, and influence cytokine expression and cellular transformation in experimental models. The tissue distribution and cellular localization of the different AU-rich binding proteins (AUBPs), however, have not been uniformly characterized in the mouse, a model for ARE mRNA decay. We therefore carried out immunoblot and immunohistochemical analyses of the different AUBPs using the same mouse tissues. We show that HuR protein, a major AUBP that stabilizes the ARE mRNAs, is most strongly expressed in the thymus, spleen (predominantly in lymphocytic cells), intestine, and testes. AUF1 protein, a negative regulator of ARE mRNA stability, displayed strong expression in thymus and spleen cells within lymphocytic cells, moderate expression in the epithelial linings of lungs, gonadal tissues, and nuclei of most neurons in the brain, and little expression in the other tissues. Tristetraprolin, a negative regulator of ARE mRNA stability, displayed a largely non-overlapping tissue distribution with AUF1 and was predominantly expressed in the liver and testis. KH-type splicing regulatory protein, a presumptive negative regulator of ARE mRNA stability, was distributed widely in murine organs. These results indicate that HuR and AUF1, which functionally oppose each other, have generally similar distributions, suggesting that the balance between HuR and AUF1 is likely important in control of short lived mRNA degradation, lymphocyte development, and/or cytokine production, and possibly in certain aspects of neurological function.     

Correlation between intrinsic mRNA stability and the affinity of AUF1 (huRNP D) and HuR for A+U-rich mRNAs

Presence of A+U-rich elements (AREs) within 3-untranslated regions (3UTRs) of numerous mRNAs has been associated with rapid mRNA turnover; however, the interaction of specific factors with AREs is also associated with mRNA stabilization. Recently, two ARE binding proteins with putative mRNA destabilizing (AUF1) and stabilizing (HuR) properties have been described. However, no direct comparison of AUF1 and HuR binding properties has been made. Therefore, we examined the relative affinities of p37AUF1 and HuR for a diverse set of ARE-containing mRNAs encoding -adrenergic receptors, a proto-oncogene, and a cytokine. We find that high-affinity AUF1 binding appears to require elements beyond primary nucleotide sequence. In contrast, binding of HuR appears considerably less constrained. As a functional correlate, we determined the ability of these specific mRNA sequences to affect the stability of chimeric -globin mRNA constructs. Although the relative affinity of AUF1 and HuR are generally predictive of mRNA stability, we find that certain mRNA sequences do not conform to these generalizations.     

Identification and characterization of proteins that selectively interact with isoforms of the mRNA binding protein AUF1 (hnRNP D)

The mRNAs that encode certain cytokines and proto-oncogenes frequently contain a typical AU-rich motif that is located in their 3'-untranslated region. The protein AUF1 is the first factor identified that binds to AU-rich regions and mediates the fast degradation of the target mRNAs. AUF1 exists as four different isoforms (p37, p40, p42 and p45) that are generated by alternative splicing. The fact that AUF1 does not degrade mRNA itself had led to the suggestion that other AUF1 interacting proteins might be involved in the process of selective mRNA degradation. Here we used the yeast two-hybrid system in order to identify proteins that bind to AUF1. We detected AUF1 itself, as well as the ubiquitin-conjugating enzyme E2I and three RNA binding proteins: NSEP-1, NSAP-1 and IMP-2, as AUF1 interacting proteins. We confirmed all interactions in vitro and mapped the protein domains that are involved in the interaction with AUF1. Gel-shift assays with the recombinant purified proteins suggest that the interacting proteins and AUF1 can bind simultaneously to an AU-rich RNA oligonucleotide. Most interestingly, the AUF1 interacting protein NSEP-1 showed an endoribonuclease activity in vitro. These data suggest the possibility that the identified AUF1 interacting proteins might be involved in the regulation of mRNA stability mediated by AUF1.     

Thermodynamics and kinetics of Hsp70 association with A + U-rich mRNA-destabilizing sequences

Rapid mRNA degradation directed by A + U-rich elements (AREs) is mediated by the interaction of specific RNA-binding proteins to these sequences. The protein chaperone Hsp70 has been identified in a cellular complex containing the ARE-binding protein AUF1 and has also been detected in direct contact with A + U-rich RNA substrates, indicating that Hsp70 may be involved in the regulation of ARE-directed mRNA turnover. By using gel mobility shift and fluorescence anisotropy assays, we have determined that Hsp70 directly and specifically associates with U-rich RNA substrates in solution. With the ARE from tumor necrosis factor alpha (TNFalpha) mRNA, Hsp70 forms a dynamic complex consistent with a 1:1 association of protein:RNA but demonstrates cooperative binding behavior on polyuridylate substrates. Unlike AUF1, the RNA binding activity of Hsp70 is not regulated by ion-dependent folding of the TNFalpha ARE, suggesting that AUF1 and Hsp70 recognize distinct binding determinants on this RNA substrate. Binding of Hsp70 to the TNFalpha ARE is driven entirely by enthalpy at physiological temperatures, indicating that burial of hydrophobic surfaces is likely the principal mechanism stabilizing the Hsp70.RNA complex. Potential roles for the interaction of Hsp70 with ARE-containing mRNAs in the regulation of mRNA turnover and/or translational efficiency are discussed.     

Chaperone Hsp27, a novel subunit of AUF1 protein complexes, functions in AU-rich element-mediated mRNA decay

Controlled, transient cytokine production by monocytes depends heavily upon rapid mRNA degradation, conferred by 3' untranslated region-localized AU-rich elements (AREs) that associate with RNA-binding proteins. The ARE-binding protein AUF1 forms a complex with cap-dependent translation initiation factors and heat shock proteins to attract the mRNA degradation machinery. We refer to this protein assembly as the AUF1- and signal transduction-regulated complex, ASTRC. Rapid degradation of ARE-bearing mRNAs (ARE-mRNAs) requires ubiquitination of AUF1 and its destruction by proteasomes. Activation of monocytes by adhesion to capillary endothelium at sites of tissue damage and subsequent proinflammatory cytokine induction are prominent features of inflammation, and ARE-mRNA stabilization plays a critical role in the induction process. Here, we demonstrate activation-induced subunit rearrangements within ASTRC and identify chaperone Hsp27 as a novel subunit that is itself an ARE-binding protein essential for rapid ARE-mRNA degradation. As Hsp27 has well-characterized roles in protein ubiquitination as well as in adhesion-induced cytoskeletal remodeling and cell motility, its association with ASTRC may provide a sensing mechanism to couple proinflammatory cytokine induction with monocyte adhesion and motility.