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EPSTEIN BARR VIRUS ENCODED Microrna from the BHRF1- CLUSTER IS INVOLVED in VIRAL SUPPRESSION DURING LATENCY

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EPSTEIN BARR VIRUS ENCODED Microrna from the BHRF1- CLUSTER IS INVOLVED in VIRAL SUPPRESSION DURING LATENCY EPSTEIN BARR VIRUS ENCODED microRNA FROM THE BHRF1- CLUSTER IS INVOLVED IN VIRAL SUPPRESSION DURING LATENCY N. Aizenberg1, M. Greenberg1, O. Carmi1, E. Meiri2, A. Levi2, Z. Bentwich1,2, Y. Shemer Avni1 1Department of Virology, Ben Gurion University of the Negev, Beer-Sheva, Israel, 2Rosetta Genomics, Rehovot, Israel Epstein Barr Virus (EBV) is a large DNA virus of the Herpes family that infects normal human B cells, is the etiologic agent of Infectious Mononucleosis and is strongly associated with Burkitt’s lymphoma and, nasopharyngeal carcinoma. The virus encodes several distinct microRNAs (miRNAs) in latently infected cells, that are located in two main clusters. The BART miRNAs are expressed at high levels in stages I and II of latency, whereas the BHRF1 miRNAs are expressed at high levels in stage III latency, while being essentially undetectable in other stages of virus life cycle. Induction of lytic EBV replication in B95-8 cells, by Cisplatin and 12-O- tetradecanonoyl phorbol-13-acetate (TPA) with n-Butyric acid or by Tet on/off regulated expression of lytic-switch transactivator immediate early (IE) gene ZEBRA (BZLF1), resulted in high expression of one of the BHRF-1- microRNA-cluster. This cluster is localized on the 5' and 3'-UTRs of BHRF1 mRNA. EBV replication resulted in increase of LMP-1 (latency associated protein 1) and viral load (determined by FACS and RT-PCR). BHRF-1, a viral homologue to Bcl2 oncogene, is also a member of the IE proteins of EBV. Antisense blockage of BHRF1-miRNA expression resulted in increased number of viral particles, up to ten fold, in comparison to control nonspecific antisense treatment. Taken together, these results support the notion that microRNAs play a central role in the control of viral life cycle and that their suppression leads to lytic activation and escape from latency. These findings also indicate the potential use of microRNAs as bases for antiviral therapies. TRANSCRIPTION REGULATION OF microRNA GENES N. Bar, R. Dikstein Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel MicroRNAs (miRNAs) is a class of functional non-coding RNA molecules, about 18- 22 nucleotides in length, which serve as gene regulators. MiRNAs usually have a specific expression pattern, indicating tight regulation that is probably at the transcriptional level. The primary miRNA transcript goes through several steps of processing, eventually producing the mature form, which act to downregulate translation and mRNA stability of the target genes. Although a subset of miRNA genes was shown to be transcribed by RNA polymerase II, not much is known about the transcription regulation of these genes. It seems likely that miRNA promoters contain specific features, which will help to regulate their transcription and mediate the recruitment of the processing machinery. My goal is to characterize the structural and functional features of miRNA promoters using computational and experimental approaches. We determined the transcription start sites (TSS) of two independently transcribed miRNAs, miR22 and the polycistronic transcript of miR1-2 and miR133a- 1. We found that miR22 promoter acts exclusively via RNA pol II machinery. We further analyzed the promoter and found three important regions. Two regions distal from the TSS contain enhancers and a third element situated at -75 to -65 relative to the TSS seems to be a novel core promoter element. The promoter of miR1-2, 133a-1 shows complex behavior: under basal conditions it is transcribed by RNA pol III and is controlled by important upstream elements including Oct-1 binding site, and consensus motifs surrounding the TSSs. Upon induction of muscle differentiation the same promoter is occupied and transcribed by RNA pol II resulting in high level of miRNA production. Our work reveals a remarkable complexity of miRNA transcription. When more miRNA promoters will be validated and analyzed, it will be interesting to examine our findings in a wider context. THE RNAi/microRNA PATHWAY INVOLVEMENT IN GUT IMMUNITY HOMEOSTASIS M. Biton, A. Levin, M. Slyper, E. Pikarsky, Y. Ben-Neriah Department of Immunology, Lautenberg Center, Hebrew University - Hadassah Medical School, Jerusalem, Israel The discovery of the RNAi/microRNA pathway a few years ago opened up a new dimension in the area of post-transcriptional regulation. One of the first thoughts was that cellular RNAi was designed to protect cells against exogenous nucleic acids introduced by pathogens. In mammals, the epithelial barrier of the intestinal tract regenerates every 5 days. This lining may be constantly eroded by pathogens and other substances of the gut lumen, making the intestinal epithelium an interesting model for exploring the role of the RNAi/microRNA pathway in gut protection and maintenance of homeostasis. Recent studies suggest an intensive crosstalk between the gut epithelial cells and the innate immune system and several mechanisms have been proposed to explain the purpose of this crosstalk. One possibility, linked to the original postulate on the role of RNAi is that the pathway plays a role in recruiting the innate immune system to protect the gut against invaders. To address this possibility we created a conditional knockout of the RNaseIII, DICER gene in gut epithelium. It appears that these mice are more vulnerable to pathogen invasion and suffer from chronic gut inflammation. The gut epithelium is composed of four distinct cell types: enterocytes, enteroendocrine, Paneth and goblet cells. Both goblet and paneth cells play a role in the gut innate immune system and a delicate equilibrium of these cells governs the gut homeostasis. Conditional dicer deletion in gut epithelial cells results in goblet cell reduction and Paneth cell maturation defect in the large and the small intestine, respectively. We are studying two possibilities, that the failed immune status of dicer-deficient mice is linked to dicer-regulated developmental effects in the epithelium, or alternatively, dicer-linked immune changes affect epithelial differentiation. In any case our findings imply a relationship between gut epithelial cells and the immune system that is controlled by the RNAi/microRNA pathway. REGULATION OF micro-RNA EXPRESSION BY THE p53 TUMOR SUPPRESSOR R. Brosh, A. Liran, N. Goldfinger, V. Rotter Department of Molecular and Cellular Biology, Weizmann Institute of Science, Rehovot, Israel microRNAs are small RNAs that regulate gene expression at the post-translational level either by translational inhibition or by mRNA cleavage. These novel RNA species were recently implicated in a variety of cancer types and were shown to be capable of acting both as tumor suppressors and oncogenes. We intend to utilize our well-characterized in-vitro model of cellular transformation to identify signatures of microRNA expression associated with distinct steps of malignant progression and with defined genetic alterations such as p53 inactivation, INK4A locus silencing and H-Ras activation. Identification and characterization of transformation-related microRNA expression signatures may help in the development of prognostic and therapeutic applications. microRNA EXPRESSION ASSOCIATED WITH ESTROGEN SUGGESTS A ROLE FOR microRNAs IN FISH VITELLOGENESIS A. Cohen1,2, M. Shmoish3, L. Levi1,2, U. Cheruti1, B. Sivan2, E. Lubzens1 1Israel Oceanographic and Limnological Research, Haifa, Israel, 2Hebrew University of Jerusalem, Rehovot, Israel, 3Technion-Israel Institute of Technology, Haifa, Israel MicroRNAs (miRNAs) are non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. To date, no comprehensive research was conducted on the involvement of miRNAs in hormonal regulated processes, such as the formation of oviparous eggs in fish by a process known as vitellogenesis. 17-beta estradiol (E2) is a key hormone in the regulation of vitellogenesis in non-mammalian vertebrates, including teleosts. E2 regulates, through estrogen receptor 1 (Esr1), the synthesis of vitellogenins (Vtgs) in the liver of females but also the liver of E2 treated males responds by synthesizing Vtgs and other proteins associated with vitellogenesis. Microarrays of miRNAs were hybridized using RNA from whole body homogenates of male ZF, exposed to E2 for 0, 1, 4, 12 and 24 hours. In addition, changes in miRNA expression profiles in livers of vitellogenic vs. non-vitellogenic female ZF were elucidated and the expression of conserved miRNAs was analyzed by real time RT-PCR in livers of vitellogenic vs. non-vitellogenic females of the marine fish gilthead seabream (GSB) to show conserved patterns. Microarray results for ZF revealed 38 miRNAs that were differentially expressed after exposure to E2, of which 25 miRNAs were mostly changed 12 hours after treatment, coinciding with the expression of Esr1 and Vtg3. The most up-regulated miRNA, miR-196b and its precursors showed similar tissue specific expression patterns after treatment. MiRNA specific expression patterns were found in the livers of vitellogenic vs. non- vitellogenic females and some of them showed a conserved pattern between ZF and GSB. We show here the first example for estrogen regulation of miRNAs and for the possible implication of miRNAs in estrogen-regulated processes such as vitellogenesis. Our results suggest a novel pathway for the pleiotropic effects of estrogens and can also have impact on other related research fields such as endocrine disrupting chemicals and cancer research. HOST-PATHOGEN CROSS-TALK VIA microRNA N. Elefant1,
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