Beta-adrenergic agonists that down-regulate receptor mRNA up-regulate a M(r) 35,000 protein(s) that selectively binds to beta-adrenergic receptor mRNAs.

In an effort to explore the molecular basis for agonist-induced destabilization of beta-adrenergic receptor mRNA, we investigated the nature of RNA-binding proteins both in untreated and agonist-treated DDT1-MF2 smooth muscle cells. Messenger RNAs for the alpha 1b-, beta 1-, and beta 2-adrenergic receptors as well as for beta-globin were transcribed in vitro, incubated with cytosolic fractions, covalently cross-linked by short-wave UV light, and analyzed by SDS-polyacrylamide gel electrophoresis. A prominent M(r) 35,000 radiolabeled protein(s) with the following characteristics was identified: (i) binds selectively to beta 1- and beta 2-adrenergic receptor mRNAs, both of which undergo agonist-induced down-regulation; (ii) does not bind to either alpha 1b-adrenergic receptor mRNA, which does not undergo agonist induced down-regulation, or to beta-globin mRNA; (iii) displays binding to beta 2-adrenergic receptor mRNA that is selectively competed by poly(U) RNA, but not poly(A), -(C), or -(G) RNA; and (iv) displays binding to receptor mRNA that can be competed by RNA harboring destabilizer sequences that are AU-rich and AUUUA pentamer-rich. The abundance of the M(r) 35,000 RNA-binding protein selective for beta-adrenergic receptor message, a factor we term beta ARB protein, varies inversely with the level of receptor mRNA, being induced by agonists that down-regulate receptor mRNA.     

Translational control of beta2-adrenergic receptor mRNA by T-cell-restricted intracellular antigen-related protein

Cellular expression of the beta(2)-adrenergic receptor (beta(2)-AR) is suppressed at the translational level by 3'-untranslated region (UTR) sequences. To test the possible role of 3'-UTR-binding proteins in translational suppression of beta(2)-AR mRNA, we expressed the full-length 3'-UTR or the adenylate/uridylate-rich (A+U-rich element (ARE)) RNA from the 3'-UTR sequences of beta(2)-AR in cell lines that endogenously express this receptor. Reversal of beta(2)-adrenergic receptor translational repression by retroviral expression of 3'-UTR sequences suggested that ARE RNA-binding proteins are involved in translational suppression of beta(2)-adrenergic receptor expression. Using a 20-nucleotide ARE RNA from the receptor 3'-UTR as an affinity ligand, we purified the proteins that bind to these sequences. T-cell-restricted intracellular antigen-related protein (TIAR) was one of the strongly bound proteins identified by this method. UV-catalyzed cross-linking experiments using in vitro transcribed 3'-UTR RNA and glutathione S-transferase-TIAR demonstrated multiple binding sites for this protein on beta(2)-AR 3'-UTR sequences. The distal 340-nucleotide region of the 3'-UTR was identified as a target RNA motif for TIAR binding by both RNA gel shift analysis and immunoprecipitation experiments. Overexpression of TIAR resulted in suppression of receptor protein synthesis and a significant shift in endogenously expressed beta(2)-AR mRNA toward low molecular weight fractions in sucrose gradient polysome fractionation. Taken together, our results provide the first evidence for translational control of beta(2)-AR mRNA by TIAR.     

CA repeats in the 3'-untranslated region of bcl-2 mRNA mediate constitutive decay of bcl-2 mRNA

An AU-rich element (ARE) in the 3'-untranslated region (UTR) of bcl-2 mRNA has previously been shown to be responsible for destabilizing bcl-2 mRNA during apoptosis through increasing AUF1 binding. In the present study, we investigated the effect of the region upstream of the ARE on bcl-2 mRNA stability using serial deletion constructs of the 3'-UTR of bcl-2. Deletion of 30 nucleotides mostly consisting of the CA repeats, located upstream of the ARE, resulted in the stabilization of bcl-2 mRNA abundance, in the absence or presence of the ARE. The specificity of the CA repeats in terms of destabilizing bcl-2 mRNA was proven by the substituting the CA repeats with other alternative repeats of purine/pyrimidine, but this had no effect on the stability of bcl-2 mRNA. CA repeats alone, however, failed to confer instability to bcl-2 or gfp reporter mRNAs, indicating a requirement for additional sequences in the upstream region of the 3'-UTR. Serial deletion and replacement of a part of the region upstream of the CA repeats revealed that the entire 131-nucleotide upstream region is an essential prerequisite for the CA repeat-dependent destabilization of bcl-2 mRNA. Unlike the ARE, CA repeat-mediated degradation of bcl-2 mRNA was not accelerated upon apoptotic stimulus. Moreover, the upstream sequences and CA repeats are conserved among mammals. Collectively, CA repeats contribute to the constitutive decay of bcl-2 mRNA in the steady states, thereby maintaining appropriate bcl-2 levels in mammalian cells.     

Distinct domains of AU-rich elements exert different functions in mRNA destabilization and stabilization by p38 mitogen-activated protein kinase or HuR

AU-rich elements (AREs) control the expression of numerous genes by accelerating the decay of their mRNAs. Rapid decay and deadenylation of beta-globin mRNA containing AU-rich 3' untranslated regions of the chemoattractant cytokine interleukin-8 (IL-8) are strongly attenuated by activating the p38 mitogen-activated protein (MAP) kinase/MAP kinase-activated protein kinase 2 (MK2) pathway. Further evidence for a crucial role of the poly(A) tail is provided by the loss of destabilization and kinase-induced stabilization in ARE RNAs expressed as nonadenylated forms by introducing a histone stem-loop sequence. The minimal regulatory element in the IL-8 mRNA is located in a 60-nucleotide evolutionarily conserved sequence with a structurally and functionally bipartite character: a core domain with four AUUUA motifs and limited destabilizing function on its own and an auxiliary domain that markedly enhances destabilization exerted by the core domain and thus is essential for the rapid removal of RNA targets. A similar bipartite structure and function are observed for the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE. Stabilization in response to p38/MK2 activation is seen with the core domain alone and also after mutation of the AUUUA motifs in the complete IL-8 ARE. Stabilization by ARE binding protein HuR requires different sequence elements. Binding but no stabilization is observed with the IL-8 ARE. Responsiveness to HuR is gained by exchanging the auxiliary domain of the IL-8 ARE with that of GM-CSF or with a domain of the c-fos ARE, which results in even stronger responsiveness. These results show that distinct ARE domains differ in function with regard to destabilization, stabilization by p38/MK2 activation, and stabilization by HuR.     

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.     

Dicer and positive charge of proteins decrease the stability of RNA containing the AU-rich element of GM-CSF

AU-rich elements (AREs) in the 3'-untranslated region of mRNAs promote rapid decay of the mRNAs for certain cytokines, including that encoding granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that an RNA molecule based on the ARE of GM-CSF mRNA is cleaved between U and A residues in the presence of bovine serum albumin of which cleavage effect is attenuated by acetylation. Furthermore, the expression of RNA molecule containing the ARE of GM-CSF mRNA in human cell lines was increased by inhibition of histone deacetylase activity and attenuation of Dicer expression. These findings suggest that degradation of mRNAs containing an ARE might be regulated by positive charge of polypeptides and Dicer.     

Interplay of two functionally and structurally distinct domains of the c-fos AU-rich element specifies its mRNA-destabilizing function.

AU-rich elements (ARE) in the 3' untranslated region of many highly labile mRNAs for proto-oncogenes, lymphokines, and cytokines can act as an RNA-destabilizing element. The absence of a clear understanding of the key sequence and structural features of the ARE that are required for its destabilizing function has precluded the further elucidation of its mode of action and the basis of its specificity. Combining extensive mutagenesis of the c-fos ARE with in vivo analysis of mRNA stability, we were able to identify mutations that exhibited kinetic phenotypes consistent with the biphasic decay characteristic of a two-step mechanism: accelerated poly(A) shortening and subsequent decay of the transcribed portion of the mRNA. These mutations, which affected either an individual step or both steps, all changed the mRNA stability. Our experiments further revealed the existence of two structurally distinct and functionally interdependent domains that constitute the c-fos ARE. Domain I, which is located within the 5' 49-nucleotide segment of the ARE and contains the three AUUUA motifs, can function as an RNA destabilizer by itself. It forms the essential core unit necessary for the ARE-destabilizing function. Domain II is a 20-nucleotide U-rich sequence which is located within the 3' part of the c-fos ARE. Although it alone can not act as an RNA destabilizer, this domain serves two critical roles: (i) its presence enhances the destabilizing ability of domain I by accelerating the deadenylation step, and (ii) it has a novel capacity of buffering decay-impeding effects exerted by mutations introduced within domain I. A model is proposed to explain how these critical structural features may be involved in the c-fos ARE-directed mRNA decay pathway. These findings have important implications for furthering our understanding of the molecular basis of differential mRNA decay mediated by different AREs.     

Selective binding of ligands to beta 1, beta 2 or chimeric beta 1/beta 2-adrenergic receptors involves multiple subsites.

The molecular basis of ligand binding selectivity to beta-adrenergic receptor subtypes was investigated by designing chimeric beta 1/beta 2-adrenergic receptors. These molecules consisted of a set of reciprocal constructions, obtained by the exchange between the wild-type receptor genes of one to three unmodified transmembrane regions, together with their extracellular flanking regions. Eight different chimeras were expressed in Escherichia coli and studied with selective beta-adrenergic ligands. The evaluation of the relative effect of each chimeric exchange on ligand binding affinity was based on the analysis of delta G values, calculated from the equilibrium binding constants, as a function of the number of substituted beta 2-adrenergic receptor transmembrane domains. The data showed that the contribution of each exchanged region to subtype selectivity varies with each ligand; moreover, while several regions are critical for the pharmacological selectivity of all ligands, others are involved in the selectivity of only some compounds. The selectivity displayed by beta-adrenergic compounds towards beta 1 or beta 2 receptor subtypes thus results from a particular combination of interactions between each ligand and each of the subsites, variably distributed over the seven transmembrane regions of the receptor; these subsites are presumably defined by the individual structural properties of the ligands.     

Purification and characterization of beta-adrenergic receptor mRNA-binding proteins

Beta-adrenergic receptors (beta-ARs), like other G-protein-coupled receptors, can undergo post-transciptional regulation at the level of mRNA stability. In particular, the human beta(1)- and beta(2)-ARs and the hamster beta(2)-AR mRNA undergo beta-agonist-mediated destabilization. By UV cross-linking, we have previously described an approximately M(r) 36,000 mRNA-binding protein, betaARB, that binds to A/C+U-rich nucleotide regions within 3'-untranslated regions. Further, we have demonstrated previously that betaARB is immunologically distinct from AUF1/heterogeneous nuclear ribonucleoprotein (hnRNP) D, another mRNA-binding protein associated with destabilization of A+U-rich mRNAs (Pende, A., Tremmel, K. D., DeMaria, C. T., Blaxall, B. C., Minobe, W., Sherman, J. A., Bisognano, J., Bristow, M. R., Brewer, G., and Port, J. D. (1996) J. Biol. Chem. 271, 8493-8501). In this report, we describe the peptide composition of betaARB. Mass spectrometric analysis of an approximately M(r) 36,000 band isolated from ribosomal salt wash proteins revealed the presence of two mRNA-binding proteins, hnRNP A1, and the elav-like protein, HuR, both of which are known to bind to A+U-rich nucleotide regions. By immunoprecipitation, HuR appears to be the biologically dominant RNA binding component of betaARB. Although hnRNP A1 and HuR can both be immunoprecipitated from ribosomal salt wash proteins, the composition of betaARB (HuR alone versus HuR and hnRNP A1) appears to be dependent on the mRNA probe used. The exact role of HuR and hnRNP A1 in the regulation of beta-AR mRNA stability remains to be determined.     

In vivo studies of translational repression mediated by the granulocyte-macrophage colony-stimulating factor AU-rich element

The AU-rich element (ARE) controls the turnover of many unstable mRNAs and their translation. The granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE is known to be a destabilizing element, but its role in translation remains unclear. Here we studied in vivo the role of the GM-CSF ARE on the mRNA and protein expressions of an enhanced green fluorescent protein reporter gene. The GM-CSF ARE had a repressor effect on translation independently of its effect on mRNA levels. In the context of an internal ribosome entry site, the GM-CSF ARE still repressed translation but was no longer functional as a destabilizing element. Gel retardation assays showed that poly(A)-binding protein is displaced from the poly(A) tail when the ARE is present in the 3'-untranslated region. These data suggest that the GM-CSF ARE controls translation and mRNA decay by interfering with poly(A)-binding protein-mediated mRNA circularization.