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At least 19 recordsLinked to original sources

PubMed1998

RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein.

An important paradigm for post-transcriptional regulation is the control of cytoplasmic mRNA stability mediated by AU-rich elements (AREs) in the 3' untranslated region of transcripts encoding oncoproteins, cytokines and transcription factors. While many RNA-binding proteins have been shown to bind to AREs in vitro, neither the functional consequences nor the physiological significance of their interactions are known. Here we demonstrate a role for the embryonic lethal abnormal visual (ELAV) RNA-binding protein HuR in mRNA turnover in vivo. The ELAV family of RNA-binding proteins is highly conserved in vertebrates. In humans, there are four members; HuR is expressed in all proliferating cells, whereas Hel-N1, HuC and HuD are expressed in terminally differentiated neurons. We show that elevation of cytoplasmic HuR levels inhibits c-fos ARE-mediated RNA decay but has little effect on rapid decay directed by c-jun ARE. It appears that HuR has little effect on deadenylation but delays onset of decay of the RNA body and slows down its subsequent decay. We also show that HuR can be induced to redistribute from the nucleus to the cytoplasm and that this redistribution is associated with an altered function. Modulation of the ARE-mediated decay pathway through controlling distribution of the ELAV proteins between nucleus and cytoplasm may be a mechanism by which cell growth and differentiation is regulated.

3T3 Cells↗

PubMed1991

Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development.

The embryonic lethal abnormal visual system (elav) gene of Drosophila melanogaster is required for the development and maintenance of the nervous system. Transcripts from this locus are distributed ubiquitously throughout the nervous system at all developmental stages. A product of this gene, the ELAV protein, has homology to known RNA binding proteins. The localization of the ELAV protein was studied in all developmental stages using antibodies that were generated against a hybrid protein made in Escherichia coli. In general, these data are consistent with previous results and demonstrate that (1) the ELAV protein is detected in the developing embryonic nervous system at a time coincident with the birth of the first neurons, (2) the ELAV protein is first detected in the majority of neurons of the central and peripheral nervous systems of embryos, larvae, pupae, and adults, (3) the ELAV protein appears to be localized to the nucleus, and (4) the ELAV protein is not detected in neuroblasts or identifiable glia. These data also provide new information concerning elav expression and show that (1) ELAV is not expressed in the ganglion mother cells (GMCs), (2) while the ELAV protein is localized to the nucleus, it is not uniformly distributed throughout this structure, and (3) other Drosophila species do express an ELAV-like antigen. We propose that the elav gene provides a neuronal-housekeeping function that is required for the successful posttranscriptional processing of transcripts from a set of genes the function of which is required for proper neuronal development and maintenance.

Animals↗

PubMed1998

Messenger ribonucleoprotein complexes containing human ELAV proteins: interactions with cytoskeleton and translational apparatus.

Mammalian ELAV proteins bind to polyadenylated messenger RNAs and have specificity for AU-rich sequences. Preferred binding sites in vitro include the AUUUA pentamer and related sequences present in the 3' untranslated regions of many growth regulatory mRNAs. Human ELAV (hELAV) proteins have been implicated in post-transcriptional regulation of gene expression by their effects on the stability and translatability of growth regulatory mRNAs. We have examined the intracellular localization of ELAV proteins in neurons and in tumor cells of neuronal origin using indirect immunofluorescence, confocal microscopy and biochemical separation. Mammalian neuronal ELAV proteins are found predominantly in the cytoplasm of cells in mRNP complexes termed alpha complexes which, when associated with polysomes, form large and high density ss complexes, as assayed by glycerol and accudenz gradients, respectively. Puromycin, cytochalasin or EDTA treatments disrupt beta complexes causing the release of alpha complexes, which then appear, by confocal microscopy, as large hELAV mRNP granules associated with microtubules. Association of partially purified hELAV mRNP alpha complexes with microtubules was confirmed by in vitro reconstitution assays. Furthermore, colchicine treatment of cells suggested that association of hELAV mRNP alpha complexes with microtubules is also necessary for the formation of ss complexes. Our data suggest a model in which a subset of mRNAs is associated with microtubules as ELAV mRNP particles (alpha complexes) which, in turn, associate with polysomes to form a translational apparatus (beta complex) that is, through polysomes, associated with the microfilament cytoskeletal network. hELAV proteins in these mRNP granules may affect post-transcriptional regulation of gene expression via the intracellular transport, localization and/or translation of growth regulatory mRNAs.

Cytoskeleton↗

PubMed1998

ELAV protein HuA (HuR) can redistribute between nucleus and cytoplasm and is upregulated during serum stimulation and T cell activation.

ELAV proteins are implicated in regulating the stability and translation of cytokine and growth regulatory mRNAs such as GM-CSF, IL-2, c-myc, c-fos and GLUT1 by binding to their AU-rich 3'UTRs. The tissue-specific ELAV protein HuB (aka. Hel-N1) is predominantly cytoplasmic and has been shown to stabilize GLUT1 and c-myc mRNAs and to increase their translation following ectopic expression in 3T3-L1 cells. We report that the most widely expressed mouse ELAV protein, mHuA, is predominately nuclear in cultured NIH-3T3 cells, but is localized in the cytoplasm during early G1 of the cell cycle. Therefore, much like the primarily cytoplasmic HuB, HuA becomes temporally localized in the cytoplasm where it can potentially regulate the stability or translation of bound mRNAs. Moreover, we report that stimulation of mouse spleen cells using either mitogenic or sub-mitogenic levels of anti-CD3/CD28 resulted in a dramatic increase in the level of HuA. Upregulation of HuA corresponds to previously documented increases in cytokine expression which are due to increased mRNA stability following T cell activation. Consistent with these findings, HuA was down regulated in quiescent cells and upregulated in 3T3 cells following serum stimulation. The increase of murine HuA during the cell cycle closely resembles that of cyclin B1 which peaks in G2/M. Together with our earlier studies, these data indicate that mammalian ELAV proteins function during cell growth and differentiation due in part to their effects on posttranscriptional stability and translation of multiple growth regulatory mRNAs. This supports the hypothesis that ELAV proteins can function as transacting factors which affect a default pathway of mRNA degradation involved in the expression of growth regulatory proteins.

3T3 Cells↗

PubMed2005

The Xenopus ELAV protein ElrB represses Vg1 mRNA translation during oogenesis.

Xenopus laevis Vg1 mRNA undergoes both localization and translational control during oogenesis. We previously characterized a 250-nucleotide AU-rich element, the Vg1 translation element (VTE), in the 3'-untranslated region (UTR) of this mRNA that is responsible for the translational repression. UV-cross-linking and immunoprecipitation experiments, described here, revealed that the known AU-rich element binding proteins, ElrA and ElrB, and TIA-1 and TIAR interact with the VTE. The levels of these proteins during oogenesis are most consistent with a possible role for ElrB in the translational control of Vg1 mRNA, and ElrB, in contrast to TIA-1 and TIAR, is present in large RNP complexes. Immunodepletion of TIA-1 and TIAR from Xenopus translation extract confirmed that these proteins are not involved in the translational repression. Mutagenesis of a potential ElrB binding site destroyed the ability of the VTE to bind ElrB and also abolished translational repression. Moreover, multiple copies of the consensus motif both bind ElrB and support translational control. Therefore, there is a direct correlation between ElrB binding and translational repression by the Vg1 3'-UTR. In agreement with the reporter data, injection of a monoclonal antibody against ElrB into Xenopus oocytes resulted in the production of Vg1 protein, arguing for a role for the ELAV proteins in the translational repression of Vg1 mRNA during early oogenesis.

3' Untranslated Regions↗

PubMed1999

ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system.

We have developed an in vitro mRNA stability system using HeLa cell cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that reproduces regulated aspects of mRNA decay. The addition of cold poly(A) competitor RNA activated both a sequence-specific deadenylase activity in the extracts as well as a potent, ATP-dependent ribonucleolytic activity. The rates of both deadenylation and degradation were up-regulated by the presence of a variety of AU-rich elements in the body of substrate RNAs. Competition analyses demonstrated that trans-acting factors were required for RNA destabilization by AU-rich elements. The approximately 30-kD ELAV protein HuR specifically bound to RNAs containing an AU-rich element derived from the TNF-alpha mRNA in the in vitro system. Interaction of HuR with AU-rich elements, however, was not associated with RNA destabilization. Interestingly, recombinant ELAV proteins specifically stabilized deadenylated intermediates generated from the turnover of AU-rich element-containing substrate RNAs. These data suggest that mammalian ELAV proteins play a role in regulating mRNA stability by influencing the access of degradative enzymes to RNA substrates.

Adenosine Triphosphate↗

PubMed2001

The neuron-specific RNA-binding protein ELAV regulates neuroglian alternative splicing in neurons and binds directly to its pre-mRNA.

Drosophila melanogaster neural-specific protein, ELAV, has been shown to regulate the neural-specific splicing of three genes: neuroglian (nrg), erect wing, and armadillo. Alternative splicing of the nrg transcript involves alternative inclusion of a 3'-terminal exon. Here, using a minigene reporter, we show that the nrg alternatively spliced intron (nASI) has all the determinants required to recreate proper neural-specific RNA processing seen with the endogenous nrg transcript, including regulation by ELAV. An in vitro UV cross-linking assay revealed that ELAV from nuclear extracts cross-links to four distinct sites along the 3200 nucleotide long nASI; one EXS is positioned at the polypyrimidine tract of the default 3' splice site. ELAV cross-linking sites (EXSs) have in common long tracts of (U)-rich sequence rather than a precise consensus; moreover, each tract has at least two 8/10U elements; their importance is validated by mutant transgene reporter analysis. Further, we propose criteria for ELAV target sequence recognition based on the four EXSs, sites within the nASI that are (U) rich but do not cross-link with ELAV, and predicted EXSs from a phylogenetic comparison with Drosophila virilis nASI. These results suggest that ELAV regulates nrg alternative splicing by direct interaction with the nASI.

Alternative Splicing↗

PubMed1997

Association of the neuron-specific RNA binding domain-containing protein ELAV with the coiled body in Drosophila neurons.

The subcellular distribution of the Drosophila nervous system-specific RNA binding domain-containing protein ELAV was investigated using ELAV-specific antibodies and scanning confocal laser microscopy. ELAV is predominantly localized within the nucleus where it concentrates within discrete domains we describe as dots and webs. To characterize these discrete domains an analysis of Drosophila coiled bodies was initiated. The polyclonal antibody R288 raised against human coilin was used to identify coiled bodies in cells of the Drosophila larval central nervous system. Double-labeling immunohistochemistry showed that, similar to vertebrate and plant systems, small nuclear ribonucleoproteins are enriched within these structures. Further analysis of ELAV revealed that subnuclear domains enriched with this molecule localize within and close to coiled bodies and close to subnuclear domains enriched with splicing factors. A preliminary analysis aimed at defining a region within ELAV that may mediate a molecular or functional interaction important for its subnuclear localization revealed that deletion of the ELAV alanine/glutamine-rich amino-terminal auxiliary domain has no discernible effect on localization and that proteins produced from elav lethal alleles distribute normally.

Animals↗

PubMed1991

Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis.

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

Amino Acid Sequence↗

PubMed1998

Evidence for 3' untranslated region-dependent autoregulation of the Drosophila gene encoding the neuronal nuclear RNA-binding protein ELAV.

The Drosophila locus embryonic lethal abnormal visual system (elav) encodes a nuclear RNA-binding protein essential for normal neuronal differentiation and maintenance of neurons. ELAV is thought to play its role by binding to RNAs produced by other genes necessary for neuronal differentiation and consequently to affect their metabolism by an as yet unknown mechanism. ELAV structural homologues have been identified in a wide range of organisms, including humans, indicating an important conserved role for the protein. Analysis of elav germline transformants presented here shows that one copy of elav minigenes lacking a complete 3' untranslated region (3' UTR) rescues null mutations at elav, but that two copies are lethal. Additional in vivo experiments demonstrate that elav expression is regulated through the 3' UTR of the gene and indicate that this level of regulation is dependent upon ELAV itself. Because ELAV is an RNA-binding protein, the simplest model to account for these findings is that ELAV binds to the 3' UTR of its own RNA to autoregulate its expression. I discuss the implications of these results for normal elav function.

3' Untranslated Regions↗

PubMed2003

ATP potentiates interleukin-1 beta-induced MMP-9 expression in mesangial cells via recruitment of the ELAV protein HuR.

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.

Adenosine Triphosphate↗

PubMed2000

The neuron-enriched splicing pattern of Drosophila erect wing is dependent on the presence of ELAV protein.

Although the Drosophila melanogaster erect wing (ewg) gene is broadly transcribed in adults, an unusual posttranscriptional regulation involving alternative and inefficient splicing generates a 116-kDa EWG protein in neurons, while protein expression elsewhere or of other isoforms is below detection at this stage. This posttranscriptional control is important, as broad expression of EWG can be lethal. In this paper, we show that ELAV, a neuron-specific RNA binding protein, is necessary to regulate EWG protein expression in ELAV-null eye imaginal disc clones and that ELAV is sufficient for EWG expression in wing disc imaginal tissue after ectopic expression. Further, analysis of EWG expression elicited from intron-containing genomic transgenes and cDNA minitransgenes in ELAV-deficient eye discs shows that this regulation is dependent on the presence of ewg introns. Analyses of the ewg splicing patterns in wild-type and ELAV-deficient eye imaginal discs and in wild-type and ectopic ELAV-expressing wing imaginal discs, show that certain neuronal splice isoforms correspond to ELAV levels. The data presented in this paper are consistent with a mechanism in which ELAV increases the splicing efficiency of ewg transcripts in alternatively spliced regions rather than with a mechanism in which stability of specific splice forms is enhanced by ELAV. Additionally, we report that ELAV promotes a neuron-enriched splice isoform of Drosophila armadillo transcript. ELAV, however, is not involved in all neuron-enriched splice events.

Animals↗

PubMed2005

Neuronal ELAV proteins enhance mRNA stability by a PKCalpha-dependent pathway.

More than 1 in 20 human genes bear in the mRNA 3' UTR a specific motif called the adenine- and uridine-rich element (ARE), which posttranscriptionally determines its expression in response to cell environmental signals. ELAV (embryonic lethal abnormal vision) proteins are the only known ARE-binding factors that are able to stabilize the bound mRNAs, thereby positively controlling gene expression. Here, we show that in human neuroblastoma SH-SY5Y cells, neuron-specific ELAV (nELAV) proteins (HuB, HuC, and HuD) are up-regulated and redistributed by 15 min of treatment with the activators of PKC phorbol esters and bryostatin-1. PKC stimulation also induces nELAV proteins to colocalize with the translocated PKCalpha isozyme preferentially on the cytoskeleton, with a concomitant increase of nELAV threonine phosphorylation. The same treatment promotes stabilization of growth-associated protein 43 (GAP-43) mRNA, a well known nELAV target, and induces an early increase in GAP-43 protein concentration, again only in the cytoskeletal cell fraction. Genetic or pharmacological inactivation of PKCalpha abolishes nELAV protein cytoskeletal up-regulation, GAP-43 mRNA stabilization, and GAP-43 protein increase, demonstrating the primary role of this specific PKC isozyme in the cascade of nELAV recruitment. Finally, in vivo PKC activation is associated with an up-regulation of nELAV proteins in the hippocampal rat brain. These findings suggest a model for gene expression regulation by nELAV proteins through a PKCalpha-dependent pathway that is relevant for the cellular programs in which ARE-mediated control plays a pivotal role.

3' Untranslated Regions↗

PubMed2006

The Drosophila RNA-binding protein ELAV is required for commissural axon midline crossing via control of commissureless mRNA expression in neurons.

Drosophila ELAV is the founding member of an evolutionarily conserved family of RNA-binding proteins considered as key inducers of neuronal differentiation. Although several ELAV-specific targets have been identified, little is known about the role of elav during neural development. Here, we report a detailed characterization of the elav mutant commissural phenotype. The reduced number of commissures in elav mutant embryos is not due to loss or misspecification of neural cells but results from defects in commissural axon projections across the midline. We establish a causal relationship between the elav mutant commissural phenotype and a reduction in the expression of commissureless, a key component of the Robo/Slit growth cone repulsive signalling pathway. In the nerve cord of elav mutant embryos, comm mRNA expression is strongly reduced in neurons, but not in midline glial cells. Furthermore, specific expression of an elav transgene in posterior neurons of each segment of an elav mutant nerve cord restores comm mRNA expression in these cells, as well as the formation of posterior commissures. Finally, forced expression of comm in specific commissural neuron subsets rescues the midline crossing defects of these neurons in elav mutant embryos, further indicating that elav acts cell autonomously on comm expression.

Animals↗

PubMed2005

Shared RNA-binding sites for interacting members of the Drosophila ELAV family of neuronal proteins.

The product of the Drosophila embryonic lethal abnormal visual system is a conserved protein (ELAV) necessary for normal neuronal differentiation and maintenance. It possesses three RNA-binding domains and is involved in the regulation of RNA metabolism. The long elav 3'-untranslated region (3'-UTR) is necessary for autoregulation. We used RNA-binding assays and in vitro selection to identify the ELAV best binding site in the elav 3'-UTR. This site resembles ELAV-binding sites identified previously in heterologous targets, both for its nucleotide sequence and its significant affinity for ELAV (K(d) 40 nM). This finding supports our model that elav autoregulation depends upon direct interaction between ELAV and elav RNA. We narrowed down the best binding site to a 20 nt long sequence A(U5)A(U3)G(U2)A(U6) in an alternative 3' exon. We propose and test a model in which the regulated use of this alternative 3' exon is involved in normal elav regulation. Found in NEurons (FNE), another neuronal RNA-binding protein paralogous to ELAV, also binds this site. These observations provide a molecular basis for the in vivo interactions reported previously between elav and fne.

3' Untranslated Regions↗

PubMed2000

Function of RRM domains of Drosophila melanogaster ELAV: Rnp1 mutations and rrm domain replacements with ELAV family proteins and SXL.

Members of the ELAV family of proteins contain three RNA recognition motifs (RRMs), which are highly conserved. ELAV, a Drosophila melanogaster member of this family, provides a vital function and exhibits a predominantly nuclear localization. To investigate if the RNA-binding property of each of the ELAV RRMs is required for ELAV's in vivo function, amino acid residues critical in RNA binding for each RRM were individually mutated. A stringent genetic complementation test revealed that when the mutant protein was the sole source of ELAV, RNA-binding ability of each RRM was essential to ELAV function. To assess the degree to which each domain was specific for ELAV function and which domains perhaps performed a function common to related ELAV proteins, we substituted an ELAV RRM with the corresponding RRM from RBP9, the D. melanogaster protein most homologous to ELAV; HuD, a human ELAV family protein; and SXL, which, although evolutionarily related, is not an ELAV family member. This analysis revealed that RRM3 replacements were fully functional, but RRM1 and RRM2 replacements were largely nonfunctional. Under less stringent conditions RRM1 and RRM2 replacements from SXL and RRM1 replacement from RBP9 were able to provide supplemental function in the presence of a mutant hypomorphic ELAV protein.

Amino Acid Motifs↗

PubMed1996

Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein.

The neuronal-specific Elav-like proteins (HuD, Hel-N, and HuC) contain three RNP-type concensus motifs and bind to AU-rich elements. We have identified and cloned a fourth member of this family (HuR) that is expressed in a wide variety of cell types. The purified recombinant protein binds avidly to the AU-rich element in c-fos and interleukin-3 mRNAs. In the case of the c-fos AU-rich element, HuR binds to a core element of 27 nucleotides that contain AUUUA, AUUUUA, and AUUUUUA motifs. Mutational analysis has shown that all three AU motifs are required for maximal binding.

Amino Acid Sequence↗

PubMed1999

Analysis of the RNA recognition motifs of human neuronal ELAV-like proteins in binding to a cytokine mRNA.

Human neuronal Elav-like proteins contain three RNP-type RNA recognition motifs (RRMs). Previous reports demonstrated that a single RRM of the proteins is not sufficient to bind to the uridine-rich stretch in the 3' untranslated region of mRNAs and that the bi-RRM peptide consisting of the first two RRMs is necessary for the binding. The present study was designed to examine the potential contributions of the first two RRMs when binding to a cytokine mRNA. Deletions of the internal or terminal amino acid residues of the first RRM (RRM1) of the HuC/ple21 ELAV-like protein completely abolished RNA binding. However, removal of any region of the second RRM (RRM2) except for the eight amino acid residues, which correspond to the potent fourth beta-sheet structure of RRM2, did not affect RNA binding. Conjugation of the eight amino acid residues to RRM1 enhanced the RNA binding as well as the entire RRM2, indicating that the octapeptide of RRM2 can be compensated for by the binding function of RRM2. The present study also showed that the substitutions of glutamic acid at 42 for aspartic acid and leucine at 44 for phenylalanine in the first potent alpha-helix structure of RRM1, as were seen in another ELAV-like protein Hel-N1, markedly affected the RNA binding.

3' Untranslated Regions↗