A common polymorphism in serotonin receptor 1B mRNA moderates regulation by miR-96 and associates with aggressive human behaviors.

Non-coding regulatory elements can transduce the human genome's response to environmental stimuli. Thus, there is a possibility that variation in non-coding regulatory elements may underlie some of the diversity in human behavior. However, this idea has remained largely untested due to the difficulty in accurately identifying regulatory elements in the 98% of the human genome that does not encode protein. The recent recognition that small trans-acting RNAs anneal to mRNA and regulate gene expression provides a means to identify and test such variants. Here, we show that microRNA-directed silencing of mRNA can be attenuated by a common human polymorphism. We have identified an element (A-element) within serotonin receptor 1B (HTR1B) mRNA that confers repression by miR-96. The repressive activity of this element is attenuated by a common human variant (G-element) that disrupts a nucleotide critical for its interaction with miR-96. Because deletion of the HTR1B gene leads to an aggressive phenotype in mice, we hypothesized an association between the A/G polymorphism and aggressive phenotypes in a sample of 359 college students. As predicted, individuals homozygous for the ancestral A-element reported more conduct-disorder behaviors than individuals with the G-element. Our studies suggest that such functional variants may be common and may help to refine the search for genes involved in complex behavioral disorders.

Polymorphism in the 3'-untranslated region of TNFalpha mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFalpha mRNA.

Tumor necrosis factor alpha (TNFalpha) acts as a beneficial mediator in the process of host defence. In recent years major interest has focused on the AU-rich elements (AREs) present in the 3'-untranslated region (3'-UTR) of TNFalpha mRNA as this region plays a pivotal role in post-transcriptional control of TNFalpha production. Certain stimuli, such as lipopolysaccharides, a component of the Gram-negative bacterial cell wall, have the ability to relinquish the translational suppression of TNFalpha mRNA imposed by these AREs in macrophages, thereby enabling the efficient production of the TNFalpha. In this study we show that the polymorphism (GAU trinucleotide insertional mutation) present in the regulatory 3'-UTR of TNFalpha mRNA of NZW mice results in the hindered binding of RNA-binding proteins, thereby leading to a significantly reduced production of TNFalpha protein. We also show that the binding of macrophage proteins to the main ARE is also decreased by another trinucleotide (CAU) insertion in the TNFalpha 3'-UTR. One of the proteins affected by the GAU trinucleotide insertional mutation was identified as HuR, a nucleo-cytoplasmic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing AREs. Since binding of this protein most likely modulates the stability, translational efficiency and transport of TNFalpha mRNA, these results suggest that mutations in the ARE of TNFalpha mRNA decrease the production of TNFalpha protein in macrophages by hindering the binding of HuR to the ARE.

The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells.

The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.

Overexpression of HuD, but not of its truncated form HuD I+II, promotes GAP-43 gene expression and neurite outgrowth in PC12 cells in the absence of nerve growth factor.

We have previously shown that the RNA-binding protein HuD binds to a regulatory element in the growth-associated protein (GAP)-43 mRNA and that this interaction involves its first two RNA recognition motifs (RRMs). In this study, we investigated the functional significance of this interaction by overexpression of human HuD protein (pcHuD) or its truncated form lacking the third RRM (pcHuD I+II) in PC12 cells. Morphological analysis revealed that pcHuD cells extended short neurites containing GAP-43-positive growth cones in the absence of nerve growth factor (NGF). These processes also contained tubulin and F-actin filaments but were not stained with antibodies against neurofilament M protein. In correlation with this phenotype, pcHuD cells contained higher levels of GAP-43 without changes in levels of other NGF-induced proteins, such as SNAP-25 and tau. In mRNA decay studies, HuD stabilized the GAP-43 mRNA, whereas HuD I+II did not have any effect either on GAP-43 mRNA stability or on the levels of GAP-43 protein. Likewise, pcHuD I+II cells showed no spontaneous neurite outgrowth and deficient outgrowth in response to NGF. Our results indicate that HuD is sufficient to increase GAP-43 gene expression and neurite outgrowth in the absence of NGF and that the third RRM in the protein is critical for this function.

The identification of an endonuclease that cleaves within an HuR binding site in mRNA.

Messenger RNAs (mRNAs) that contain U-rich elements are targeted for rapid decay. Selective inhibition of this decay results in a rapid increase in steady state level. Thus, this is an important regulatory step in gene expression. Previously, we have found that these mRNAs are selectively stabilized by a specific mRNA binding protein called HuR. The mechanism of action of HuR is not well understood. It has been postulated that HuR stabilizes mRNA by the displacement or inhibition of factors that specifically cleave or deadenyl-ate these mRNAs. In this paper, we report the identification and characterization of a novel endo-nuclease that cleaves within an HuR binding site in p27kip1 mRNA. The specificity of this endonuclease and its inhibition by HuR argue for it playing a role in the postranscriptional regulation of gene expression.

Complex contribution of the 3'-untranslated region to the expressional regulation of the human inducible nitric-oxide synthase gene. Involvement of the RNA-binding protein HuR.

Cytokine stimulation of human DLD-1 cells resulted in a marked expression of nitric-oxide synthase (NOS) II mRNA and protein accompanied by only a moderate increase in transcriptional activity. Also, there was a basal transcription of the NOS II gene, which did not result in measurable NOS II expression. The 3'-untranslated region (3'-UTR) of the NOS II mRNA contains four AUUUA motifs and one AUUUUA motif, known to destabilize the mRNAs of proto-oncogenes, nuclear transcription factors, and cytokines. Luciferase reporter gene constructs containing the NOS II 3'-UTR showed a significantly reduced luciferase activity. The embryonic lethal abnormal vision (ELAV)-like protein HuR was found to bind with high affinity to the adenylate/uridylate-rich elements of the NOS II 3'-UTR. Inhibition of HuR with antisense constructs reduced the cytokine-induced NOS II mRNA, whereas overexpression of HuR potentiated the cytokine-induced NOS II expression. This provides evidence that NOS II expression is regulated at the transcriptional and post-transcriptional level. Binding of HuR to the 3'-UTR of the NOS II mRNA seems to play an essential role in the stabilization of this mRNA.

HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation.

Colorectal carcinoma RKO cells expressing reduced levels of the RNA-binding protein HuR (ASHuR) displayed markedly reduced growth. In synchronous RKO populations, HuR was almost exclusively nuclear during early G(1), increasing in the cytoplasm during late G(1), S and G(2). The expression and half-life of mRNAs encoding cyclins A and B1 similarly increased during S and G(2), then declined, indicating that mRNA stabilization contributed to their cell cycle-regulated expression. In gel-shift assays using radiolabeled cyclin RNA transcripts and RKO protein extracts, only those transcripts corresponding to the 3'-untranslated regions of cyclins A and B1 formed RNA-protein complexes in a cell cycle-dependent fashion. HuR directly bound mRNAs encoding cyclins A and B1, as anti-HuR antibodies supershifted such RNA-protein complexes. Importantly, the expression and half-life of mRNAs encoding cyclins A and B1 were reduced in ASHuR RKO cells. Our results indicate that HuR may play a critical role in cell proliferation, at least in part by mediating cell cycle-dependent stabilization of mRNAs encoding cyclins A and B1.

Tumor antigen HuR binds specifically to one of five protein-binding segments in the 3'-untranslated region of the neurofibromin messenger RNA.

3'-untranslated regions of various mRNAs have been shown to contain sequence motifs which control mRNA stability, translatability, and efficiency of translation as well as intracellular localization. We aimed to identify protein binding regions of the long and highly conserved 3'UTR of the mRNA coding for neurofibromin, a well-known tumor suppressor protein, whose genetic deficiency causes the autosomal dominant disease neurofibromatosis type 1 (NF1). We discovered five RNA fragments that were able to undergo specific binding to proteins from cell lysates (NF1-PBRs, NF1-protein-binding regions). Additionally we identified the Elav-like protein HuR binding to NF1-PBR1. HuR interacts with AU-rich elements in the 3'UTR of many protooncogenes, cytokines, and transcription factors, thereby regulating the expression of these mRNAs on the posttranscriptional level. Transfection assays with a CAT reporter construct revealed reduced expression of the reporter, suggesting that HuR may be involved in the fine-tuning of the expression of the NF1 gene.

HuR regulates p21 mRNA stabilization by UV light.

Expression of the cyclin-dependent kinase inhibitor p21 is highly induced by many stresses, including exposure to short-wavelength UV light (UVC), which increases p21 mRNA stability. Investigation into the mechanisms underlying this stabilization process revealed that proteins present in cytoplasmic lysates of human RKO colorectal carcinoma cells formed complexes with p21 mRNA that were inducible by treatment with UVC and other stress agents. The ubiquitous Elav-type RNA-binding protein HuR was identified within the p21 mRNA-protein complexes, as antibodies recognizing HuR supershifted these complexes and revealed HuR-immunoreactive proteins complexing with p21 mRNA on Western blots. Lowering of endogenous HuR levels through expression of antisense HuR decreased p21 RNA-protein complexes, greatly reduced the UVC inducibility and half-life of p21 mRNA, and prevented UVC-mediated induction of luciferase activity in p21 3' untranslated region-containing reporter constructs. Our findings indicate that HuR plays a major role in regulating stress-induced p21 expression by enhancing p21 mRNA stability and that these effects are coupled to HuR's elevated presence in the cytoplasm.

Embryonic lethal abnormal vision-like RNA-binding proteins regulate neurite outgrowth and tau expression in PC12 cells.

The embryonic lethal abnormal vision (ELAV)-like proteins are mRNA-binding proteins that regulate mRNA stability. The neuronal members of this family are required for neuronal differentiation. We identified the binding region of purified HuD protein to a target neuronal mRNA encoding for the tau microtubule-associated protein and demonstrated an in vivo interaction between the ELAV-like protein and its target tau mRNA. We show that treatment of neuronal cells with antisense oligodeoxynucleotides directed against HuD blocks the induction of neurite outgrowth and decreases the levels of tau mRNAs, indicating that the ELAV-like proteins are required for neuronal differentiation.

The inhibitory activity of the AU-rich RNA element in the human papillomavirus type 1 late 3' untranslated region correlates with its affinity for the elav-like HuR protein.

A 57-nucleotide adenosine- and uridine-rich RNA instability element in the human papillomavirus type 1 late 3' untranslated region termed h1ARE has previously been shown to interact specifically with three nuclear proteins that failed to bind to an inactive mutant RNA. Two of those were identified as the heterogeneous ribonucleoproteins C1 and C2, whereas the third, a 38-kDa, poly(U) binding protein (p38), remained unidentified. Here we show that partially purified p38 reacts with a monoclonal antibody raised against the recently identified elav-like HuR protein, indicating that p38 is the HuR protein. Indeed, recombinant glutathione S-transferase (GST)-HuR protein binds specifically to sites within the h1ARE. Determination of the apparent Kd value of GST-HuR for the h1ARE and the inactive mutant thereof revealed that GST-HuR bound with a more than 50-fold-higher affinity to the wild-type sequence. Therefore, the binding affinity of GST-HuR for the wild-type and mutant h1AREs correlates with their inhibitory activities in transfected cells, strongly suggesting that the HuR protein is involved in the posttranscriptional regulation of human papillomavirus type 1 late-gene expression.

p21(waf1) mRNA contains a conserved element in its 3'-untranslated region that is bound by the Elav-like mRNA-stabilizing proteins.

The Elav-like proteins are specific mRNA-binding proteins that regulate mRNA stability. The neuronal members of this family (HuD, HuC, and Hel-N1) are required for neuronal differentiation. In this report, using purified HuD protein we have localized a high affinity HuD binding site to a 42-nucleotide region within a U-rich tract in the 3'-untranslated region p21(waf1) mRNA. The binding of HuD to this site is readily displaced by an RNA oligonucleotide encoding the HuD binding site of c-fos. The sequence of this binding site is well conserved in human, mouse, and rat p21(waf1) mRNA. p21(waf1) is an inhibitor of cyclin-dependent kinases and proliferating cell nuclear antigen and induces cell cycle arrest at G1/S, a requisite early step in cell differentiation. The identification of an Elav-like protein binding site in the 3'-untranslated region of p21(waf1) provides a novel link between the induction of differentiation, mRNA stability, and the termination of the cell cycle.

Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose expression is dramatically induced by hypoxia due in large part to an increase in the stability of its mRNA. Here we show that HuR binds with high affinity and specificity to the element that regulates VEGF mRNA stability by hypoxia. Inhibition of HuR expression abrogates the hypoxia-mediated increase in VEGF mRNA stability. Overexpression of HuR increases the stability of VEGF mRNA. However, this only occurs efficiently in hypoxic cells. We further show that the stabilization of VEGF mRNA can be recapitulated in vitro. Using an S-100 extract, we show that the addition of recombinant HuR stabilizes VEGF mRNA markedly. These data support the critical role of HuR in mediating the hypoxic stabilization of VEGF mRNA by hypoxia.

HuR, a novel target of anti-Hu antibodies, is expressed in non-neural tissues.

Paraneoplastic encephalomyelitis (PEM) is characterized by a diverse set of clinical signs that are limited to the nervous system. The serologic hallmark of PEM is the presence of circulating autoantibodies, collectively referred to as 'anti-Hu,' which immunoreact specifically with members of the Elav protein family. Until recently, the ELAV antigens were only detected in neurons, thus strongly supporting a role for anti-Hu antibodies in the selective neural tissue injury in PEM. The identification of HuR, however, a new member with a broad, non-neural pattern of RNA expression, raises several fundamental questions regarding PEM. First, why are non-neural tissues spared in PEM? Second, why is PEM predominantly associated with neuroendocrine tumors? To begin addressing these questions, we sought to determine whether the antibody response to HuR differs from the neural-specific counterparts in patients with PEM, and to characterize the protein expression pattern of this novel antigen in peripheral tissues and tumors. Using sera from 11 patients with Hu-positive PEM, we found that the majority of samples (73%) were weakly or non-reactive for recombinant HuR on Western blot, in contrast to consistently strong immunoreactivity with the neural-specific members HuD and Hel-N1. We also demonstrate that HuR is expressed at the protein level in both non-neural tissues and non-neuroendocrine tumors. These findings suggest that immunoreactive differences among Elav family members may contribute to the neural-restrictive pattern of tissue injury in patients with PEM.

The Elav-like proteins bind to AU-rich elements and to the poly(A) tail of mRNA.

The Elav-like proteins are specific mRNA binding proteins which are required for cellular differentiation. They contain three characteristic RNP2/RNP1-type RNA binding motifs. Previously we have shown that the first and second RNA binding domains bind to AU-rich elements in the 3'-UTR of mRNA. In this paper we show that the Elav-like proteins exhibit poly(A) binding activity. This activity is distinct from poly(A) binding activities that have been previously described. The Elav-like proteins specifically bind to long chain poly(A) tails. We have shown that the third RNA binding domain encompasses this poly(A) binding activity. Using poly(A)-Sepharose beads in a 'sandwich' assay we have shown that the Elav-like proteins can bind simultaneously to the AU-rich element and to the poly(A) tail.

The Elav-like proteins bind to a conserved regulatory element in the 3'-untranslated region of GAP-43 mRNA.

Previous studies have identified three brain proteins (40, 65 and 95 kDa, respectively) that specifically bind to the 3'-untranslated region of GAP-43 mRNA. In this study, using a specific monoclonal antibody, we now show that the 40-kDa proteins are members of the Elav-like protein family. This family of specific RNA-binding proteins comprise three neural specific members called HuD, HuC, and Hel-N1. We have shown that purified recombinant HuD can bind with high affinity to GAP-43 mRNA. In addition, we have mapped the binding site to a highly conserved 26-nucleotide sequence within the regulatory element. The binding of HuD to this site is readily displaced by RNA oligonucleotides encoding other HuD binding sites. We also show that only the first and second RNA binding domains of HuD are required for selective binding to GAP-43 mRNA.

Localization of the human HuR gene to chromosome 19p13.2.

The HuR gene encodes a specific RNA binding protein that is a member of the human Elav-like gene family. This family of proteins, which includes HuD, HuC and Hel-N1, is involved in cellular differentiation. Alterations of HuD and Hel-N1 structure are associated with small cell lung tumors and medulloblastomas. To investigate a possible linkage of the HuR gene to malignancy, the locus of the gene was mapped on human metaphase chromosomes. Analysis of the fluorescence signals on banded chromosomes showed that the HuR gene is localized to human chromosome 19p13.2 the cell cycle and proliferation (Levine et al. 1993; Gao et al. 1994; Liu et al. 1995; Chung et al. 1996). HuD and Hel-N1 are aberrantly spliced in human tumors yielding isoforms that are incapable of inducing differentiation (Gao et al. 1994; Antic et al. 1996). Thus it is feasible that rearrangements of the HuR gene may occur in other human tumors. In order to investigate this possibility we have mapped the HuR gene using fluorescence in-situ hybridization (FISH).

Purification and properties of HuD, a neuronal RNA-binding protein.

HuD is a human neuronal specific RNA-binding protein. In this study we have purified HuD and examined its RNA binding properties in detail. HuD binds to mRNAs that contain an AU-rich element with high affinity. In the case of the c-fos AU-rich element, HuD binds to a 27-nucleotide core element comprising AUUUA, AUUUUA, and AUUUUUA motifs. Mutation in any two of these motifs abrogates binding. HuD contains two tandem RNA recognition motifs (RRM), a basic domain, and a third RRM. Deletion analysis has shown that only the first and second RRMs are essential for RNA binding. Thus, these specific RNA binding properties support the idea that the HuD regulates gene expression at the posttranscriptional level.

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.