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Oz Solomon,
Shirley Oren, Michal Safran,
Naamit Deshet-Unger,
Pinchas Akiva,
Jasmine Jacob-Hirsch,
Karen Cesarkas,
Reut Kabesa,
Ninette Amariglio,
Ron Unger,
Gideon Rechavi,
Eran Eyal
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ABSTRACT: Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.
RNA 03/2013; · 5.09 Impact Factor
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ABSTRACT: Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1-p110 isoform. We were unable to achieve overexpression of ADAR1-p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24-48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development.
PLoS ONE 01/2012; 7(7):e41576. · 4.09 Impact Factor
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ABSTRACT: Adenosine to inosine (A-to-I) RNA-editing is an essential post-transcriptional mechanism that occurs in numerous sites in the human transcriptome, mainly within Alu repeats. It has been shown to have consistent levels of editing across individuals in a few targets in the human brain and altered in several human pathologies. However, the variability across human individuals of editing levels in other tissues has not been studied so far.
Here, we analyzed 32 skin samples, looking at A-to-I editing level in three genes within coding sequences and in the Alu repeats of six different genes. We observed highly consistent editing levels across different individuals as well as across tissues, not only in coding targets but, surprisingly, also in the non evolutionary conserved Alu repeats.
Our findings suggest that A-to-I RNA-editing of Alu elements is a tightly regulated process and, as such, might have been recruited in the course of primate evolution for post-transcriptional regulatory mechanisms.
BMC Genomics 10/2010; 11:608. · 4.07 Impact Factor
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Sivan Osenberg,
Nurit Paz Yaacov, Michal Safran,
Sharon Moshkovitz,
Ronit Shtrichman,
Ofra Sherf,
Jasmine Jacob-Hirsch,
Gilmor Keshet,
Ninette Amariglio,
Joseph Itskovitz-Eldor,
Gideon Rechavi
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ABSTRACT: Adenosine to Inosine (A-to-I) RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA) protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3'UTRs, 5'UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs). We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, we speculate that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions.
PLoS ONE 01/2010; 5(6):e11173. · 4.09 Impact Factor
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Lavinia Nardinocchi,
Valentina Pantisano,
Rosa Puca,
Manuela Porru,
Aurora Aiello,
Annalisa Grasselli,
Carlo Leonetti, Michal Safran,
Gideon Rechavi,
David Givol,
Antonella Farsetti,
Gabriella D'Orazi
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ABSTRACT: Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the "angiogenic switch" during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.
Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.
These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.
PLoS ONE 01/2010; 5(12):e15048. · 4.09 Impact Factor
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ABSTRACT: A-to-I RNA editing is essential for the development of normal cells and is involved in a wide variety of biological pathways. Currently, limited information suggests linkage between changes in RNA editing levels and the development of cancer. We aimed to explore the possible linkage between altered RNA editing levels and the development of human urinary bladder neoplasms.
Thirty-two patients underwent transurethral resection of bladder tumor. Normal and tumoral urinary bladder tissues were obtained from each patient during surgery. Total RNA was extracted from tissue cells and converted by RT-PCR reaction to cDNA molecules for further analysis. We explored known editing sites in RNA encoding for proteins (BLCAP, Cyfip2, FLNA, GluB Q/R) as well as in RNA transcribed from Alu elements in noncoding regions of the genes encoding for CARD11, FANCC, MDM4, BRCA1, and RBBP9 proteins. Editing levels were determined using Sequenom MassARRAY Compact Analyzer.
Eleven tumoral tissues obtained were low grade TCC, 14 high grade TCC, 1 CIS, and another 5 inflammation. One sample contained only normal tissue. We got a total number of 30 normal bladder tissue samples and overall 29 paired samples (i.e., normal and tumoral tissues obtained from the same patient). Statistical analysis revealed no significant changes in editing levels between normal and tumoral tissues.
Relying on the results obtained for 9 different editing sites, it can be determined that RNA editing is an epigenetic mechanism that does not participate in the evolution of urinary bladder cancer.
Urologic Oncology 02/2009; 29(1):21-6. · 3.22 Impact Factor
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Nurit Paz,
Erez Y Levanon,
Ninette Amariglio,
Amy B Heimberger,
Zvi Ram,
Shlomi Constantini,
Zohar S Barbash,
Konstantin Adamsky, Michal Safran,
Avi Hirschberg,
Meir Krupsky,
Issachar Ben-Dov,
Simona Cazacu,
Tom Mikkelsen,
Chaya Brodie,
Eli Eisenberg,
Gideon Rechavi
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ABSTRACT: Adenosine-to-inosine (A-to-I) RNA editing was recently shown to be abundant in the human transcriptome, affecting thousands of genes. Employing a bioinformatic approach, we identified significant global hypoediting of Alu repetitive elements in brain, prostate, lung, kidney, and testis tumors. Experimental validation confirmed this finding, showing significantly reduced editing in Alu sequences within MED13 transcripts in brain tissues. Looking at editing of specific recoding and noncoding sites, including in cancer-related genes, a more complex picture emerged, with a gene-specific editing pattern in tumors vs. normal tissues. Additionally, we found reduced RNA levels of all three editing mediating enzymes, ADAR, ADARB1, and ADARB2, in brain tumors. The reduction of ADARB2 correlated with the grade of malignancy of glioblastoma multiforme, the most aggressive of brain tumors, displaying a 99% decrease in ADARB2 RNA levels. Consistently, overexpression of ADAR and ADARB1 in the U87 glioblastoma multiforme cell line resulted in decreased proliferation rate, suggesting that reduced A-to-I editing in brain tumors is involved in the pathogenesis of cancer. Altered epigenetic control was recently shown to play a central role in oncogenesis. We suggest that A-to-I RNA editing may serve as an additional epigenetic mechanism relevant to cancer development and progression.
Genome Research 12/2007; 17(11):1586-95. · 13.61 Impact Factor
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Ronit Pasvolsky,
Sara W Feigelson,
Sara Sebnem Kilic,
Amos J Simon,
Guy Tal-Lapidot,
Valentin Grabovsky,
Jill R Crittenden,
Ninette Amariglio, Michal Safran,
Ann M Graybiel,
Gideon Rechavi,
Shifra Ben-Dor,
Amos Etzioni,
Ronen Alon
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ABSTRACT: Leukocyte and platelet integrins rapidly alter their affinity and adhesiveness in response to various activation (inside-out) signals. A rare leukocyte adhesion deficiency (LAD), LAD-III, is associated with severe defects in leukocyte and platelet integrin activation. We report two new LAD cases in which lymphocytes, neutrophils, and platelets share severe defects in beta(1), beta(2), and beta(3) integrin activation. Patients were both homozygous for a splice junction mutation in their CalDAG-GEFI gene, which is a key Rap-1/2 guanine exchange factor (GEF). Both mRNA and protein levels of the GEF were diminished in LAD lymphocytes, neutrophils, and platelets. Consequently, LAD-III platelets failed to aggregate because of an impaired alpha(IIb)beta(3) activation by key agonists. beta(2) integrins on LAD-III neutrophils were unable to mediate leukocyte arrest on TNFalpha-stimulated endothelium, despite normal selectin-mediated rolling. In situ subsecond activation of neutrophil beta(2) integrin adhesiveness by surface-bound chemoattractants and of primary T lymphocyte LFA-1 by the CXCL12 chemokine was abolished. Chemokine inside-out signals also failed to stimulate lymphocyte LFA-1 extension and high affinity epitopes. Chemokine-triggered VLA-4 adhesiveness in T lymphocytes was partially defective as well. These studies identify CalDAG-GEFI as a critical regulator of inside-out integrin activation in human T lymphocytes, neutrophils, and platelets.
Journal of Experimental Medicine 08/2007; 204(7):1571-82. · 13.85 Impact Factor
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ABSTRACT: Fibroblast growth factor-2 (basic FGF), a potent inducer of angiogenesis, and the naphthalene sulfonic distamycin A derivative, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino])-bis-(1,3-naphtalene disulfonate) (PNU145156E), which exhibits in vivo antiangiogenic activity, form a tight reversible (1:1) complex. PNU145156E binds to the heparin and the selenate-binding sites on bFGF. The cis bFGF-heparin (2:1) complex, essential for the activation of the angiogenic process, is thus prevented. The nature of the forces involved in bFGF:PNU145156E complex, using the wild-type and the K128Q, K138Q, K134Q, and K128Q-K138Q point mutated bFGFs was sought. Based on thermodynamic analysis of the complexation constants, protein temperature stability profiles by ultraviolet absorption, circular dichroism measurements, fluorescence Förster energy-transfer, and anisotropy studies, in harmony with the published x-ray crystallographic structure, the following molecular interactions are proposed: reduced coulombic interactions, hence loosening of the complex by the removal of charged polar groups from the bFGF-heparin binding cleft resulted in decreased binding constants and in a change in the binding mode from polar to nonpolar. Concomitantly, upon mutation, the protein was rendered more compact, less flexible, and less aqueously exposed compared with the wild type. These were further pronounced with the double mutant: weaker dominantly nonpolar protein-drug interactions were accompanied by conspicuous folding. With heparin, however, wild-type bFGF forms a tighter complex with a more compact structure.
Biophysical Journal 06/2002; 82(5):2652-64. · 3.65 Impact Factor
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ABSTRACT: One of the key events in post-angioplasty restenosis is the migration and proliferation of medial smooth muscle cells leading to neo-intima formation. This phase is mediated by several growth factors, mainly platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF2/bFGF) and heparin-binding epidermal growth factor (HB-EGF). In this study, we have focused on the role of FGF2, which requires heparan sulfate proteoglycans (HSPG) as cofactors for binding and activation of its cell surface tyrosine kinase receptor. The aim of this study was to identify and explore the effect of novel FGF antagonists on vascular smooth muscle cell (VSMC) proliferation.
We have recently identified a novel class of small, positively charged molecules sharing a porphyrin core as inhibitors of FGF2 and vascular endothelial growth factor (VEGF) activity. Here we investigated the inhibitory effect of these compounds on VSMC proliferation and their effect on heparin-induced FGF receptor activity.
We found that these molecules exert a marked inhibitory effect on FGF2-mediated smooth muscle cell (SMC) proliferation, manifested by reduced cell growth and DNA synthesis, which occurred in a dose-dependent manner with an IC(50) of approximately 1 microM of inhibitor. We demonstrate that the molecule, 5, 10, 15, 20-tetrakis (methyl-4-pyridyl)-21H, 23H-porphine tetra-p-tosylate salt (TMPP), inhibits binding of radiolabeled FGF2 to SMCs and to soluble FGF receptor 1 (FGFR1) in a manner that interferes with both ligand and receptor interactions with heparin, thereby blocking growth factor mediated SMC proliferation.
We have identified an FGF antagonist, which may serve in clinical practice as a preventive measure of restenosis.
Cardiovascular Research 02/2002; 53(1):232-41. · 6.06 Impact Factor
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ABSTRACT: Solid tumors require the formation of a vascular network derived from host blood vessels to support their growth. The heparin-binding growth factor family was the first class of angiogenesis factors to be studied. These proteins play key roles in a variety of crucial biological activities that require cell growth, differentiation, migration and chemotaxis. Our work presents the study of a group of polyanionic compounds, the naphthalene sulfonic distamycin A derivatives, named suradistas, which represents a new class of inhibitors of neo-angiogenesis that can counteract vascularization of solid tumors. By a combination of in vitro and in vivo approaches, a leader compound, PNU145156E, was selected. The results suggest that PNU145156E interacts directly with the heparin-binding growth factor bFGF in a specific manner. It forms a tight but reversible 1 to 1 complex with the protein, inducing conformational changes, which render bFGF less stable, preventing the interaction with heparin and the biologically effective dimerization of the growth factor.
Journal of Molecular Structure.
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ABSTRACT: A survey of defined species of cell surface and extracellular matrix heparan sulfate proteoglycans (HSPG) was performed in a search for cellular proteoglycans that can promote bFGF receptor binding and biological activity. Of the various affinity-purified HSPGs tested, perlecan, the large basement membrane HSPG, is found to induce high affinity binding of bFGF both to cells deficient in HS and to soluble FGF receptors. Heparin-dependent mitogenic activity of bFGF is strongly augmented by perlecan. Monoclonal antibodies to perlecan extract the receptor binding promoting activity from active HSPG preparations. In a rabbit ear model for in vivo angiogenesis, perlecan is a potent inducer of bFGF-mediated neovascularization. These results identify perlecan as a major candidate for a bFGF low affinity, accessory receptor and an angiogenic modulator.
Cell.
