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.     

ARED 2.0: an update of AU-rich element mRNA database

The Adenylate Uridylate (AU)-Rich Element Database, ARED-mRNA version 2.0, contains information not present in the previous ARED. This includes additional data entries, new information and links to Unigene, LocusLink, RefSeq records and mouse homologue data. An ARE consensus sequence specific to the 3'UTR is the basis of ARED that demonstrated two important findings: (i) AREs are present in a large, previously unrecognized set of human mRNAs; and (ii) ARE-mRNAs encode proteins of diverse functions which are largely involved in early and transient biological responses. In this update, we have modified the strategy for identifying ARE-mRNA in order to systematically deal with inconsistencies of molecule type and mRNA region in GenBank records. Potential uses for the ARED in functional genomics are also given. The database is accessible via the web, http://rc.kfshrc.edu.sa/ared, with a new querying system that allows searching ARE-mRNAs by any public database identifier or name. The ARED website also contains relevant links to uses for the ARED.     

AU-rich transient response transcripts in the human genome: expressed sequence tag clustering and gene discovery approach

Transient response genes regulate critical biological responses that include cell proliferation, signal transduction events, and responses to exogenous agents such as inflammatory stimuli, microbes, and radiation. An important feature that ensures a timely response is the short half-life of the messenger RNA (mRNA), which is thought to be predominantly mediated by adenylate uridylate-rich sequence elements (AREs) in the 3' untranslated region (3' UTR). The repertoire and extent of transient response genes in the human genome are not known. We used a computational approach to delineate those genes that code for transient ARE mRNAs. We utilized a 3' UTR-specific ARE motif to retrieve and cluster 3'-end ESTs using a refined extraction protocol. With the availability of the entire human genome, we were able to utilize ARE EST clusters for further mining and computational prediction of ARE genes. The described approaches led to the finding of more than 1500 ARE genes in the human genome. In particular, "hidden" ARE mRNAs and alternative forms due to 3'UTR completeness, variant polyadenylation, and splicing were uncovered.     

Structural basis for recognition of AU-rich element RNA by the HuD protein

Hu proteins bind to adenosine-uridine (AU)-rich elements (AREs) in the 3' untranslated regions of many short-lived mRNAs, thereby stabilizing them. Here we report the crystal structures of the first two RNA recognition motif (RRM) domains of the HuD protein in complex with an 11-nucleotide fragment of a class I ARE (the c-fos ARE; to 1.8 A), and with an 11-nucleotide fragment of a class II ARE (the tumor necrosis factor alpha ARE; to 2.3 A). These structures reveal a consensus RNA recognition sequence that suggests a preference for pyrimidine-rich sequences and a requirement for a central uracil residue in the clustered AUUUA repeats found in class II AREs. Comparison to structures of other RRM domain-nucleic acid complexes reveals two base recognition pockets in all the structures that interact with bases using residues in conserved ribonucleoprotein motifs and at the C-terminal ends of RRM domains. Different conformations of nucleic acid can be bound by RRM domains by using different combinations of base recognition pockets and multiple RRM domains.     

Adenovirus infection induces HuR relocalization to facilitate virus replication

HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3′-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication.     

Assays for monitoring viral manipulation of host ARE-mRNA turnover

Early host responses to viral infection rapidly induce an antiviral gene expression program that limits viral replication and recruits sentinel cells of the innate immune system. These responses are mediated by cytokines. The mRNAs that encode cytokines typically harbor destabilizing adenine- and uridine-rich elements (AREs) that direct their constitutive degradation in the cytoplasm. In response to a variety of signals, including viral infection, small pools of cytoplasmic ARE-mRNAs are rapidly stabilized and translated. Thus, mRNA stability plays a key role in antiviral gene expression. Intriguingly, recent studies have identified viral proteins that specifically target ARE-mRNAs for stabilization, suggesting that certain proteins encoded by ARE-mRNAs may be advantageous for infection. Here, we discuss the development of a suite of sensitive and complementary assays to monitor ARE-mRNA turnover. These include luciferase- and destabilized-GFP-based assays that can be adapted for high-throughput screening applications.     

Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU

The AU-rich element (ARE) in the 3' untranslated region of unstable mRNAs mediate their rapid degradation. ARE binding proteins (AUBPs) have been described that either stabilize or otherwise degrade ARE-mRNAs by recruiting the exosome, a complex of 3'-to-5' exoribonucleases. We have identified RHAU, a putative DExH RNA helicase that was isolated in association with the ARE of urokinase plasminogen activator mRNA (ARE(uPA)). RHAU physically interacts with the deadenylase PARN and the human exosome and enhances the deadenylation and decay of ARE(uPA)-mRNAs. An alternatively spliced isoform of RHAU that localized to the cytoplasm had a more pronounced effect on ARE(uPA)-mRNA destabilization than full-length RHAU. Furthermore, the ATPase activity of RHAU is essential for its mRNA-destabilizing function. ARE(uPA)-mRNA recognition by RHAU may be mediated through its RNA-dependent interaction with the AUBPs HuR and NFAR1. A model is presented to describe the action of RHAU in ARE(uPA)-directed mRNA turnover.     

AU-rich element-mediated mRNA decay can occur independently of the miRNA machinery in mouse embryonic fibroblasts and Drosophila S2-cells

AU-rich elements (AREs) are regulatory sequences located in the 3' untranslated region of many short-lived mRNAs. AREs are recognized by ARE-binding proteins and cause rapid mRNA degradation. Recent reports claimed that the function of AREs may be--at least in part--relayed through the miRNA pathway. We have revisited this hypothesis using dicer knock-out mouse embryonic fibroblasts and cultured Drosophila cells. In contrast to the published results, we find no evidence for a general requirement of the miRNA pathway in the function of AREs. Endogenous ier3 mRNA, which is known to contain a functional ARE, was degraded rapidly at indistinguishable rates in wild type and dicer knock-out mouse embryonic fibroblasts. In cultured Drosophila cells, both ARE-containing GFP reporter mRNAs and the endogenous cecA1 mRNA were resistant to depletion of the mi/siRNA factors dcr-1, dcr-2, ago1 and ago2. Furthermore, the Drosophila miRNA originally proposed to recognize AU-rich elements, miR-289, is not detectably expressed in flies or cultured S2 cells. Even our attempts to overexpress this miRNA from its genomic hairpin sequence failed. Thus, this sequence cannot serve as link between the miRNA and the AU-rich element mediated silencing pathways. Taken together, our studies in mammalian and Drosophila cells strongly argue that AREs can function independently of miRNAs.     

Involvement of microRNA in AU-rich element-mediated mRNA instability

AU-rich elements (AREs) in the 3' untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-alpha. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. We further observed that miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation.     

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.     

Aberrant regulation of mRNA 3' untranslated region in cancers and inflammation

Almost 10% of mammalian coding mRNAs contain in their 3' untranslated region a sequence rich in adenine and uridine residues known as AU-rich element (ARE). Many of them encode oncogenes (for instance c-Myc and c-Fos), cell cycle regulators (cyclin D1, A1, B1), cytokines (TNFalpha, IL2) and growth factors (GM-CSF) which are overexpressed in cancer or inflammatory diseases due to increased mRNA stability and/or translation. AREs are recognized by a group of proteins, collectively called AUBPs which display various functions. For instance, HuR/ELAV is mainly known to protect ARE-containing mRNAs from degradation, while AUF1, TTP and KSRP act to destabilize their bound target mRNAs and TIA/TIAR to inhibit their translation. Alterations in ARE sequences or AUBP abundance, cellular localization or activity due to post-translational modifications such as phosphorylation can promote or enhance malignancy or perturb immune homeostasis. Here, c-myc and TNFalpha are chosen as examples to illustrate how altered 3' UTR gene regulation impacts on pathologies.     

Highly selective actions of HuR in antagonizing AU-rich element-mediated mRNA destabilization

Human RNA-binding protein HuR, a nucleocytoplasmic shuttling protein, is a ubiquitously expressed member of the family of Hu proteins, which consist of two N-terminal RNA recognition motifs (RRM1 and RRM2), a hinge region, and a C-terminal RRM (RRM3). Although in vitro experiments showed indiscriminate binding of Hu proteins synthesized in bacterial systems to many different AU-rich elements (AREs), in vivo studies have pointed to a cytoplasmic role for HuR protein in antagonizing the rapid decay of some specific ARE-containing mRNAs, depending on physiological situations. By ectopically overexpressing HuR and its mutant derivatives in NIH 3T3 cells to mimic HuR upregulation of specific ARE-containing mRNAs in other systems, we have examined the in vivo ARE-binding specificity of HuR and dissected its functionally critical domains. We show that in NIH 3T3 cells, HuR stabilizes reporter messages containing only the c-fos ARE and not other AREs. Two distinct binding sites were identified within the c-fos ARE, the 5' AUUUA-containing domain and the 3' U-stretch-containing domain. These actions of HuR are markedly different from those of another ARE-binding protein, hnRNP D (also termed AUF1), which in vivo recognizes AUUUA repeats found in cytokine AREs and can exert both stabilizing and destabilizing effects. Further experiments showed that any combination of two of the three RRM domains of HuR is sufficient for strong binding to the c-fos ARE in vitro and to exert an RNA stabilization effect in vivo comparable to that of intact HuR and that the hinge region containing nucleocytoplasmic shuttling signals is dispensable for the stabilization effect of HuR. Our data suggest that the ARE-binding specificity of HuR in vivo is modulated to interact only with and thus regulate specific AREs in a cell type- and physiological state-dependent manner.     

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.