MicroRNAs (miRNAs) have been implicated in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein–Barr virus (EBV) encodes 23 miRNAs of unknown function. Here we show that the EBV-encoded miRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. MiR-BART2 guides cleavage within the 3′-untranslated region (3′UTR) of BALF5 by virtue of its complete complementarity to its target. Induction of the lytic viral replication cycle results in a reduction of the level of miR-BART2 with a strong concomitant decrease of cleavage of the BALF5 3′UTR. Expression of miR-BART2 down-regulates the activity of a luciferase reporter gene containing the BALF5 3′UTR. Forced expression of miR-BART2 during lytic replication resulted in a 40–50% reduction of the level of BALF5 protein and a 20% reduction of the amount of virus released from EBV-infected cells. Our results are compatible with the notion that EBV-miR-BART2 inhibits transition from latent to lytic viral replication.

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3′-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3′UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3′ UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer. Mol Cancer Res; 8(4); 529-38. ©2010 AACR.

MicroRNAs (miRNAs) represent a conserved class of small noncoding RNAs that are found in all higher eukaryotes as well as some DNA viruses. miRNAs are 20 to 25 nucleotides in length and have important regulatory functions in biological processes such as embryonic development, cell differentiation, hormone secretion, and metabolism. Furthermore, miRNAs have been implicated in the pathology of various diseases, including cancer. miRNA expression profiles not only classify different types of cancer but also may even help to characterize distinct tumor stages, therefore constituting a valuable tool for prognosis. Here we report the miRNA profile of Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) tissue samples characterized by cloning and sequencing. We found that all EBV miRNAs from the BART region are expressed in NPC tissues, whereas EBV miRNAs from the BHRF1 region are not found. Moreover, we identified two novel EBV miRNA genes originating from the BART region that have not been found in other tissues or cell lines before. We also identified three new human miRNAs which might be specific for nasopharyngeal tissues. We further show that a number of different cellular miRNAs, including miR-15a and miR-16, are up-or downregulated in NPC tissues compared to control tissues. We found that the tumor suppressor BRCA-1 is a target of miR-15a as well as miR-16, suggesting a miRNA role in NPC pathogenesis.MicroRNAs (miRNAs) constitute a conserved class of endogenously expressed small noncoding RNAs of 20 to 25 nucleotides (nt) in size with regulatory functions in various cellular processes (1,4,7,14). miRNA genes are transcribed by RNA polymerase II or III as primary transcripts that are processed to stem-loop structured precursors (pre-miRNAs) by the nuclear microprocessor complex containing the RNase III Drosha and its cofactor DGCR8 (33, 51). Pre-miRNAs are transported to the cytoplasm by the export receptor exportin-5, where the RNase III Dicer cleaves off the loop of the hairpin, thereby creating a short double-stranded RNA

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The Epstein–Barr virus (EBV) is an oncogenic human Herpes virus found in ∼15% of diffuse large B-cell lymphoma (DLBCL). EBV encodes miRNAs and induces changes in the cellular miRNA profile of infected cells. MiRNAs are small, non-coding RNAs of ∼19–26 nt which suppress protein synthesis by inducing translational arrest or mRNA degradation. Here, we report a comprehensive miRNA-profiling study and show that hsa-miR-424, -223, -199a-3p, -199a-5p, -27b, -378, -26b, -23a, -23b were upregulated and hsa-miR-155, -20b, -221, -151-3p, -222, -29b/c, -106a were downregulated more than 2-fold due to EBV-infection of DLBCL. All known EBV miRNAs with the exception of the BHRF1 cluster as well as EBV-miR-BART15 and -20 were present. A computational analysis indicated potential targets such as c-MYB, LATS2, c-SKI and SIAH1. We show that c-MYB is targeted by miR-155 and miR-424, that the tumor suppressor SIAH1 is targeted by miR-424, and that c-SKI is potentially regulated by miR-155. Downregulation of SIAH1 protein in DLBCL was demonstrated by immunohistochemistry. The inhibition of SIAH1 is in line with the notion that EBV impedes various pro-apoptotic pathways during tumorigenesis. The down-modulation of the oncogenic c-MYB protein, although counter-intuitive, might be explained by its tight regulation in developmental processes.

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Here we provide evidence that EBNA2 is methylated in vivo and that methylation of EBNA2 is a prerequisite for binding to SMN. We present SMN as a novel binding partner of EBNA2 by showing that EBNA2 colocalizes with SMN in nuclear gems and that both proteins can be coimmunoprecipitated from cellular extract. Furthermore, in vitro methylation of either wild-type EBNA2 or a glutathione S-transferase-EBNA2 fusion protein encompassing the arginine-glycine (RG) repeat element is necessary for in vitro binding to the Tudor domain of SMN. The recently shown functional cooperation of SMN and EBNA2 in transcriptional activation and the previous observation of a severely reduced transformation potential yet strongly enhanced transcriptional activity of an EBNA2 mutant lacking the RG repeat indicate that binding of SMN to EBNA2 is a critical step in B-cell transformation by Epstein-Barr virus.The Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with a variety of human tumors (reviewed in reference 25). The virus transforms B cells into continuously growing lymphoblastoid cell lines by expressing 12 genes (reviewed in reference 1). The nuclear antigen EBNA2 is the first viral gene expressed after infection and is essential for EBVmediated transformation through the activation of viral and cellular genes like the viral EBNAs and the latent membrane proteins LMP1, LMP2A, and LMP2B as well as the cellular CD21, CD23, c-fgr, and c-myc (reviewed in reference 13) and AML-2 genes (26). EBNA2 does not bind directly but is tethered to promoters by interacting with cellular transcription factors like RBPJ, Sp-1/Spi-B, hnRNP-D/AUF1, or ATF/ CRE. Domains of EBNA2 critical for transformation of B cells and for activation of gene expression had been identified previously through mutational analysis (6,28,29). EBNA2 binds to RBPJ with its conserved WWP 325 (18) motif, the deletion of which results in a severe reduction of activation of the LMP1 promoter and a complete loss of transforming capacity (6). An adjacent arginine-glycine (RG) repeat element between amino acids (aa) 337 and 354 of EBNA2 was shown elsewhere to be critical but not essential for B-cell transformation in vitro, but the deletion of aa 337 to 354 increased the potential of EBNA2 to activate the LMP1 promoter (29). While most interacting partners bind to the C terminus of EBNA2, we had recently demonstrated that EBNA2 interacts through its N-terminal aa 121 to 216 with a putative helicase/ATPase termed DP103 (12), which in turn binds to SMN, the product of the spinal muscular atrophy gene (SMN) (4,5,30). The survival of motor neurons (SMN) gene is lost or mutated in spinal muscular atrophy (SMA) (17). SMN is found in different macromolecular complexes, and one of these has recently been shown to facilitate the assembly of spliceosomal U snRNPs by mediating the attachment of the Sm proteins onto snRNAs U1, U2, U4, and U5 (7,20,22). The function of the SMN complex involves direct binding of Sm proteins to SMN, an interaction that is strongly enha...