ELAV tumor antigen, Hel-N1, increases translation of neurofilament M mRNA and induces formation of neurites in human teratocarcinoma cells.
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Human ELAV proteins are implicated in cell growth and differentiation via regulation of mRNA expression in the cytoplasm. In human embryonic teratocarcinoma (hNT2) cells transfected with the human neuronal ELAV-like protein, Hel-N1, neurites formed, yet cells were not terminally differentiated. Cells in which neurite formation was associated with Hel-N1 overexpression, also expressed increased levels of endogenous neurofilament M (NF-M) protein, which distributed along the neurites. However, steady-state levels of NF-M mRNA remained similar whether or not hNT2 cells were transfected with Hel-N1. These findings suggest that turnover of NF-M mRNA was not affected by Hel-N1 expression, despite the fact that Hel-N1 can bind to the 3' UTR of NF-M mRNA and was found directly associated with NF-M mRNA in transfected cells. Analysis of the association of NF-M mRNA with the translational apparatus in Hel-N1 transfectants showed nearly complete recruitment to heavy polysomes, indicating that Hel-N1 caused an increase in translational initiation. Our results suggest that the stability and/or translation of ARE-containing mRNAs can be regulated independently by the ELAV protein, Hel-N1, depending upon sequence elements in the 3' UTRs and upon the inherent turnover rates of the mRNAs that are bound to Hel-N1 in vivo. (+info)
An AU-rich sequence in the 3'-UTR of plasminogen activator inhibitor type 2 (PAI-2) mRNA promotes PAI-2 mRNA decay and provides a binding site for nuclear HuR.
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The plasminogen activator inhibitor type 2 (PAI-2) gene is regulated by transcriptional and post-transcriptional processes. We have previously shown that insertion of the 3'-untranslated region (3'-UTR) of PAI-2 mRNA into the 3'-UTR of a beta-globin reporter mRNA reduces constitutive beta-globin mRNA expression and that this requires, at least in part, an AU-rich motif. Here we have directly assessed the role of this motif in PAI-2 mRNA stability using both chimeric and non-chimeric reporter systems. We first show that the full-length PAI-2 mRNA is indeed unstable with a half-life of 1 h. Using the c-fos promoter-driven human growth hormone (HGH) mRNA as a reporter, we demonstrate that the 580 nt 3'-UTR of PAI-2 accelerates chimeric HGH mRNA decay in a process which is dependent on the intact AU-rich sequence. Furthermore, disruption of this motif within a constitutively expressed PAI-2 cDNA produces a 2.5- and 2. 7-fold increase in PAI-2 mRNA and protein levels in HT-1080 cells, respectively. RNA electrophoretic mobility shift and supershift assays indicate that this motif provides a specific binding site for cellular proteins that include nuclear HuR. Taken together, these data show that a correlation exists between the binding of HuR to the AU-rich motif in vitro and the destabilizing properties conferred by this sequence in vivo. (+info)
Requirement of RBP9, a Drosophila Hu homolog, for regulation of cystocyte differentiation and oocyte determination during oogenesis.
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The Drosophila RNA binding protein RBP9 and its Drosophila and human homologs, ELAV and the Hu family of proteins, respectively, are highly expressed in the nuclei of neuronal cells. However, biochemical studies suggest that the Hu proteins function in the regulation of mRNA stability, which occurs in the cytoplasm. In this paper, we show that RBP9 is expressed not only in the nuclei of neuronal cells but also in the cytoplasm of cystocytes during oogenesis. Despite the predominant expression of RBP9 in nerve cells, mutational analysis revealed a female sterility phenotype rather than neuronal defects for Rbp9 mutants. The female sterility phenotype of the Rbp9 mutants resulted from defects in oogenesis; the lack of Rbp9 activity caused the germarium region of the mutants to be filled with undifferentiated cystocytes. RBP9 appears to stimulate cystocyte differentiation by regulating the expression of bag-of-marbles (bam) mRNA, which encodes a developmental regulator of germ cells. RBP9 protein bound specifically to bam mRNA in vitro, which is required for cystocyte proliferation, and the number of cells that expressed BAM protein was increased 5- to 10-fold in the germarium regions of Rbp9 mutants. These results suggest that RBP9 protein binds to bam mRNA to down regulate BAM protein expression, which is essential for the initiation of cystocyte differentiation into functional egg chambers. In hypomorphic Rbp9 mutants, cystocytes differentiated into egg chambers; however, oocyte determination and positioning were perturbed. Therefore, the concentrated localization of RBP9 protein in the oocyte of the early egg chambers may be required for proper oocyte determination or positioning. (+info)
Binding of neuronal ELAV-like proteins to the uridine-rich sequence in the 3'-untranslated region of tumor necrosis factor-alpha messenger RNA.
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Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that is involved in the pathogenesis of several human CNS disorders. The AU-rich element (ARE) in the 3'-untranslated region (UTR) of TNF-alpha mRNA is implicated in post-transcriptional control of TNF-alpha. In this study, we showed that a human neuronal ELAV-like protein binds to the ARE in the 3'-UTR of TNF-alpha mRNA. The protein binds to the uridine stretch in AUUUA pentanucleotides inside the ARE in the 3'-UTR of TNF-alpha mRNA. The TNF-alpha mRNA-binding region in the protein appears to be identical to the c-myc and IL-3 mRNA-binding regions. Moreover, this study showed that in vitro treatment of neuroblastoma cells with interleukin-4 (IL-4), which inhibits TNF-alpha production, reduced the expression of the neuronal ELAV-like proteins. These results suggest that the expression of neuronal ELAV-like proteins may be closely associated with the expression of TNF-alpha in neuronal cells. (+info)
HuD, a neuronal-specific RNA-binding protein, is a putative regulator of N-myc pre-mRNA processing/stability in malignant human neuroblasts.
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N-myc gene copy numbers and transcription rates are similar in N (neuroblastic, tumorigenic) and S (non-neuronal, non-tumorigenic) neuroblastoma cells with chromosomally integrated amplified N-myc genes. However, N cells show significantly higher N-myc mRNA levels than S cells. Therefore, post-transcriptional control of N-myc gene expression must differ between these cell types. Since no differences in N-myc mRNA half-life were found between N and S cells from two cell lines, steady-state levels of N-myc pre-mRNA processing intermediates were analysed. Results suggest that the differences in N-myc expression arise primarily at the nuclear post-transcriptional level. The neuronal-specific RNA-binding Hu proteins are present in cytoplasmic and nuclear fractions of N cells and one of them, HuD, binds specifically to both exonic and intronic N-myc RNA sequences. In sense and antisense HuD-transfected N cells, there are coordinate changes in HuD and N-myc expression levels. Thus, we propose that HuD plays a role in the nuclear processing/stability of N-myc pre-mRNA in N-type neuroblastoma cells. (+info)
Embryonic lethal abnormal vision-like RNA-binding proteins regulate neurite outgrowth and tau expression in PC12 cells.
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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. (+info)
Anti-Hu antibody titre and brain metastases before and after treatment for small cell lung cancer.
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OBJECTIVES: To follow up the level of anti-Hu antibody titres during chemotherapy and to compare the pattern of metastases and other neurological complications before and after chemotherapy in patients with small cell lung cancer (SCLC) with and without low titre anti-Hu antibodies. Seventeen per cent of patients with SCLC without paraneoplastic syndromes have a low titre of anti-Hu antibodies in their serum. Previous studies suggested that these antibodies correlate with a more indolent tumour growth. METHODS: The serum of 52 consecutive patients with SCLC were studied before and during chemotherapy, and the correlation with stage of disease and pattern of metastases was examined. All serum samples were investigated using western blot and enzyme linked immunosorbent assay (ELISA) with HuD recombinant protein. All patients with SCLC were investigated using MRI of the brain, CSF, bone marrow aspiration, ultrasound of the abdomen, and radionuclide bone scan. RESULTS: Nine (17%) of 52 SCLC serum samples were positive by western blot. At the time of diagnosis none of the anti-Hu positive patients had either CNS (brain or leptomeningeal), epidural, adrenal, or bone marrow metastases and 56% had limited disease. In eight of 43 anti-Hu negative patients CNS metastases were found at the time of diagnosis, and only 30% had limited disease. The prevalence of bone and liver metastases was similar in both groups. Survival was 11 (SD ) months for the 43 anti-Hu negative and 10 (SD 6) months for the nine anti-Hu positive patients. Male:female ratio in the anti-Hu negative group was 4.4:1, and in the anti-Hu positive group 2:1. CONCLUSIONS: No anti-Hu antibody positive serum, as tested by western blot, became negative during chemotherapy. Anti-Hu positive and anti-Hu negative patients had similar survival, but anti-Hu positive patients tended to be women, had limited disease at the time of tumour diagnosis, and initially metastases seemed to spare the nervous system. (+info)
Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and the peripheral nervous systems.
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Hu proteins are mammalian embryonic lethal abnormal visual system (ELAV)-like neuronal RNA-binding proteins that contain three RNA recognition motifs. Although Drosophila ELAV is required for the correct differentiation and survival of neurons, the roles played by the Hu genes in the mammalian nervous system remain largely unknown. To explore the in vivo functions of mouse Hu proteins, we overexpressed them in rat pheochromocytoma PC12 cells, where they induced neuronal phenotype in the absence of nerve growth factor. We have characterized the functions of various forms of mHuB and mHuC bearing point mutations or deletions. Mutants of mHuC that had amino acid exchanges in the RNP1 domain of the first or second RNA recognition motifs (RRMs) lost biologic activity as well as RNA-binding activity. In addition, the mutants containing only the third RRM failed to induce the neuronal phenotype in PC12 cells and inhibited the biologic activity of cotransfected wild-type mHuB and mHuC, thus acting as a dominant-negative form. However, these mutants could not suppress the nerve growth factor-induced differentiation of PC12 cells. Further, we misexpressed wild-type and dominant-negative Hu in E9.5 mouse embryos, by using electroporation into the neural tube at the level of the rhombencephalon. mHuB and mHuC induced the ectopic expression of neuronal markers, whereas the dominant-negative forms of mHuB and mHuC suppressed the differentiation of central nervous system motor neurons. From these results, we suggest that Hu proteins are required for neuronal differentiation in the mammalian nervous system. (+info)