Direct binding of specific AUF1 isoforms to tandem zinc finger domains of tristetraprolin (TTP) family proteins

Tristetraprolin (TTP) is the prototype of a family of CCCH tandem zinc finger proteins that can bind to AU-rich elements in mRNAs and promote their decay. TTP binds to mRNA through its central tandem zinc finger domain; it then promotes mRNA deadenylation, considered to be the rate-limiting step in eukaryotic mRNA decay. We found that TTP and its related family members could bind to certain isoforms of another AU-rich element-binding protein, HNRNPD/AUF1, as well as a related protein, laAUF1. The interaction domain within AUF1p45 appeared to be a C-terminal "GY" region, and the interaction domain within TTP was the tandem zinc finger domain. Surprisingly, binding of AUF1p45 to TTP occurred even with TTP mutants that lacked RNA binding activity. In cell extracts, binding of AUF1p45 to TTP potentiated TTP binding to ARE-containing RNA probes, as determined by RNA gel shift assays; AUF1p45 did not bind to the RNA probes under these conditions. Using purified, recombinant proteins and a synthetic RNA target in FRET assays, we demonstrated that AUF1p45, but not AUF1p37, increased TTP binding affinity for RNA ～5-fold. These data suggest that certain isoforms of AUF1 can serve as "co-activators" of TTP family protein binding to RNA. The results raise interesting questions about the ability of AUF1 isoforms to regulate the mRNA binding and decay-promoting activities of TTP and its family members as well as the ability of AUF1 proteins to serve as possible physical links between TTP and other mRNA decay proteins and structures.     

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.     

Assembly of Functional Ribonucleoprotein Complexes by AU-rich Element RNA-binding Protein 1 (AUF1) Requires Base-dependent and -independent RNA Contacts

AU-rich element RNA-binding protein 1 (AUF1) regulates the stability and/or translational efficiency of diverse mRNA targets, including many encoding products controlling the cell cycle, apoptosis, and inflammation by associating with AU-rich elements residing in their 3′-untranslated regions. Previous biochemical studies showed that optimal AUF1 binding requires 33–34 nucleotides with a strong preference for U-rich RNA despite observations that few AUF1-associated cellular mRNAs contain such extended U-rich domains. Using the smallest AUF1 isoform (p37^AUF1) as a model, we employed fluorescence anisotropy-based approaches to define thermodynamic parameters describing AUF1 ribonucleoprotein (RNP) complex formation across a panel of RNA substrates. These data demonstrated that 15 nucleotides of AU-rich sequence were sufficient to nucleate high affinity p37^AUF1 RNP complexes within a larger RNA context. In particular, p37^AUF1 binding to short AU-rich RNA targets was significantly stabilized by interactions with a 3′-purine residue and largely base-independent but non-ionic contacts 5′ of the AU-rich site. RNP stabilization by the upstream RNA domain was associated with an enhanced negative change in heat capacity consistent with conformational changes in protein and/or RNA components, and fluorescence resonance energy transfer-based assays demonstrated that these contacts were required for p37^AUF1 to remodel local RNA structure. Finally, reporter mRNAs containing minimal high affinity p37^AUF1 target sequences associated with AUF1 and were destabilized in a p37^AUF1-dependent manner in cells. These findings provide a mechanistic explanation for the diverse population of AUF1 target mRNAs but also suggest how AUF1 binding could regulate protein and/or microRNA binding events at adjacent sites.     

Destabilization of interleukin-6 mRNA requires a putative RNA stem-loop structure, an AU-rich element, and the RNA-binding protein AUF1

Interleukin-6 mRNA is unstable and degraded with a half-life of 30 min. Instability determinants can entirely be attributed to the 3' untranslated region. By grafting segments of this region to stable green fluorescent protein mRNA and subsequent scanning mutagenesis, we have identified two conserved elements, which together account for most of the instability. The first corresponds to a short noncanonical AU-rich element. The other, 80 nucleotides further 5', comprises a sequence predicted to form a stem-loop structure. Neither element alone was sufficient to confer full instability, suggesting that they might cooperate. Overexpression of myc-tagged AUF1 p37 and p42 isoforms as well as suppression of endogenous AUF1 by RNA interference stabilized interleukin-6 mRNA. Both effects required the AU-rich instability element. Similarly, the proteasome inhibitor MG132 stabilized interleukin-6 mRNA probably through an increase of AUF1 levels. The mRNA coimmunoprecipitated specifically with myc-tagged AUF1 p37 and p42 in cell extracts but only when the AU-rich instability element was present. These results indicate that AUF1 binds to the AU-rich element in vivo and promotes IL-6 mRNA degradation.     

Analysis of the structural determinants for RNA binding of the human protein AUF1/hnRNP D

The protein AUF1/hnRNP D was one of the first factors identified that binds to the AU-rich region of certain mRNAs and mediates their fast degradation. Here we describe experiments to address the structural determinants for the binding of AUF1 to the RNA by combining comparative molecular modeling with gel shift assays. From our model of the RNA binding region of AUF1 we predicted that it interacts with RNA predominantly through stacking interactions that do not provide base-specific recognition. Only two RNA positions bound by AUF1 show base preferences: one for pyrimidine bases and the second for a conserved adenine residue. Gel shift assays with a panel of RNA oligonucleotides largely confirmed these model-based binding determinants. An alignment with proteins of the hnRNP family demonstrated that the amino acids involved in the stacking interactions are conserved whereas those that confer a base-specific recognition in AUF1 are variable.     

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.     

Chaperone Hsp27 modulates AUF1 proteolysis and AU-rich element-mediated mRNA degradation

AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-α) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues--Ser(15), Ser(78), and Ser(82)-by a number of kinases, including the mitogen-activated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2-Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization.     

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.     

Identification of AUF1 (heterogeneous nuclear ribonucleoprotein D) as a component of the alpha-globin mRNA stability complex.

mRNA turnover is an important regulatory component of gene expression and is significantly influenced by ribonucleoprotein (RNP) complexes which form on the mRNA. Studies of human alpha-globin mRNA stability have identified a specific RNP complex (alpha-complex) which forms on the 3' untranslated region (3'UTR) of the mRNA and appears to regulate the erythrocyte-specific accumulation of alpha-globin mRNA. One of the protein activities in this multiprotein complex is a poly(C)-binding activity which consists of two proteins, alphaCP1 and alphaCP2. Neither of these proteins, individually or as a pair, can bind the alpha-globin 3'UTR unless they are complexed with the remaining non-poly(C) binding proteins of the alpha-complex. With the yeast two-hybrid screen, a second alpha-complex protein was identified. This protein is a member of the previously identified A+U-rich (ARE) binding/degradation factor (AUF1) family of proteins, which are also known as the heterogeneous nuclear RNP (hnRNP) D proteins. We refer to these proteins as AUF1/hnRNP-D. Thus, a protein implicated in ARE-mediated mRNA decay is also an integral component of the mRNA stabilizing alpha-complex. The interaction of AUF1/hnRNP-D is more efficient with alphaCP1 relative to alphaCP2 both in vitro and in vivo, suggesting that the alpha-complex might be dynamic rather than a fixed complex. AUF1/hnRNP-D could, therefore, be a general mRNA turnover factor involved in both stabilization and decay of mRNA.     

AUF1/hnRNP D is a novel protein partner of the EBER1 noncoding RNA of Epstein-Barr virus

Epstein-Barr virus (EBV)–infected cells express two noncoding RNAs called EBV-encoded RNA (EBER) 1 and EBER2. Despite their high abundance in the nucleus (about 10⁶ copies), the molecular function of these noncoding RNAs has remained elusive. Here, we report that the insertion into EBER1 of an RNA aptamer that binds the bacteriophage MS2 coat protein allows the isolation of EBER1 and associated protein partners. By combining MS2-mediated selection with stable isotope labeling of amino acids in cell culture (SILAC) and analysis by mass spectrometry, we identified AUF1 (AU-rich element binding factor 1)/hnRNP D (heterogeneous nuclear ribonucleoprotein D) as an interacting protein of EBER1. AUF1 exists as four isoforms generated by alternative splicing and is best known for its role in destabilizing mRNAs upon binding to AU-rich elements (AREs) in their 3′ untranslated region (UTR). Using UV crosslinking, we demonstrate that predominantly the p40 isoform of AUF1 interacts with EBER1 in vivo. Electrophoretic mobility shift assays show that EBER1 can compete for the binding of the AUF1 p40 isoform to ARE-containing RNA. Given the high abundance of EBER1 in EBV-positive cells, EBER1 may disturb the normal homeostasis between AUF1 and ARE-containing mRNAs or compete with other AUF1-interacting targets in cells latently infected by EBV.     

14-3-3sigma is a p37 AUF1-binding protein that facilitates AUF1 transport and AU-rich mRNA decay

Short-lived cytokine mRNAs contain an AU-rich destabilizing element (ARE). AUF1 proteins bind the ARE, undergo shuttling, and promote cytoplasmic ARE-mRNA decay through a poorly understood mechanism. We therefore identified AUF1-interacting proteins that may play a role in ARE-mRNA decay. We used mass-spectrometry to identify 14-3-3sigma protein as an AUF1-interacting protein. 14-3-3sigma binds selectively and strongly to p37 AUF1 and to a lesser extent to the p40 isoform, the two isoforms most strongly associated with ARE-mRNA decay, but not to the two larger isoforms, p42 and p45. The 14-3-3sigma interaction site on p37 was mapped to a region found only in the two smaller AUF1 isoforms and which overlaps a putative nuclear localization signal (NLS). Stable overexpression of 14-3-3sigma significantly increased cytoplasmic accumulation of p37 AUF1 and reduced the steady-state level and half-life of a reporter ARE-mRNA. siRNA silencing of AUF1 eliminated the effect of 14-3-3sigma overexpression. 14-3-3sigma therefore binds to p37 AUF1, retains it in the cytoplasm probably by masking its NLS, and enhances rapid turnover of ARE-mRNAs.