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MicroRNAs (miRNAs) are short 18–23 nucleotide long noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA. Our previous miRNA profiling of diffuse large B-cell lymphoma (DLBCL) revealed a mutation in the seed sequence of miR-142-3p. Further analysis now showed that miR-142 was mutated in 11 (19.64%) of the 56 DLBCL cases. Of these, one case had a mutation in both alleles, with the remainder being heterozygous. Four mutations were found in the mature miR-142-5p, four in the mature miR-142-3p, and three mutations affected the miR-142 precursor. Two mutations in the seed sequence redirected miR-142-3p to the mRNA of the transcriptional repressor ZEB2 and one of them also targeted the ZEB1 mRNA. However, the other mutations in the mature miR-142-3p did not influence either the ZEB1 or ZEB2 3′ untranslated region (3′ UTR). On the other hand, the mutations affecting the seed sequence of miR-142-3p resulted in a loss of responsiveness in the 3′ UTR of the known miR-142-3p targets RAC1 and ADCY9. In contrast to the mouse p300 gene, the human p300 gene was not found to be a target for miR-142-5p. In one case with a mutation of the precursor, we observed aberrant processing of the miR-142-5p. Our data suggest that the mutations in miR-142 probably lead to a loss rather than a gain of function. This is the first report describing mutations of a miRNA gene in a large percentage of a distinct lymphoma subtype.

The Epstein-Barr virus (EBV) growth-transforms B-lymphocytes. The virus-encoded nuclear antigen 2 (EBNA2) is essential for transformation and activates gene expression by association with DNA-bound transcription factors such as RBPJkappa (CSL/CBF1). We have previously shown that EBNA2 contains symmetrically dimethylated Arginine (sDMA) residues. Deletion of the RG-repeat results in a reduced ability of the virus to immortalise B-cells. We now show that the RG repeat also contains asymmetrically dimethylated Arginines (aDMA) but neither non-methylated (NMA) Arginines nor citrulline residues. We demonstrate that only aDMA-containing EBNA2 is found in a complex with DNA-bound RBPJkappa in vitro and preferentially associates with the EBNA2-responsive EBV C, LMP1 and LMP2A promoters in vivo. Inhibition of methylation in EBV-infected cells results in reduced expression of the EBNA2-regulated viral gene LMP1, providing additional evidence that methylation is a prerequisite for DNA-binding by EBNA2 via association with the transcription factor RBPJkappa.

PLoS one 7(8), e42106 (2012). doi:10.1371/journal.pone.0042106, Published by PLoS [u.a.], Lawrence, Kan.

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. ISSN:1362-4962 ISSN:0301-5610

MicroRNAs (miRNAs) are involved in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein-Barr Virus (EBV) infection induces cellular non-coding (nc)RNAs e.g., the "vault" RNAs or miRNAs such as miR-21, miR-155 or miR-146a. MiR-146a is upregulated in various tumours and plays a role in innate immunity. We show that the EBV-encoded latent membrane protein 1 (LMP1) induces the expression of miR-146a via NFkappaB. LMP1 activates the miR-146a promoter but not a promoter with a mutation of the NFkappaB-response elements. Conversely, a LMP1-mutant deficient in NFkappaB-activation failed to activate the promoter. The "CAO"-LMP1 variant which has an increased potential to induce NFkappaB also showed a higher ability to activate the miR-146a promoter as compared to standard B95.8-LMP1. Northern blotting revealed high levels of miR-146a and miR-155 in the Burkitt's lymphoma cell line Jijoye which expresses LMP1 while the LMP1-deficient P3HR1 mutant derived from Jijoye expresses less miR-146a or miR-155. Likewise, EBV-latency type I Burkitt's lymphoma cells with low LMP1 levels also contain low levels of either miR-146a or miR-155 while their levels are increased in LMP1-expressing EBV-latency type III BL cells. Expression of LMP1 in P3HR1 cells upregulates miR-146a levels. Neither miR-146a nor miR-155 are detectable in BCBL-1 cells transformed by the Kaposi-Sarcoma Herpes virus (KSHV/HHV8). It is possible that the induction of miR-146a plays a role in the induction or maintenance of EBV latency by modulating innate immune responses to the virus infected host cell.

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.

The Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to the genesis of several human lymphoproliferative diseases (for a review, see reference 33). The EBV-encoded nuclear antigen 2 (EBNA2) is a viral transactivator essential for EBV-induced transformation of resting human B lymphocytes, by promoting the expression of the transforming latent membrane proteins LMP1 and 2, the nuclear EBV Cp promoter-driven EBNA proteins, and the cellular genes CD23 and c-fgr (for review, see reference 15). EBNA2 does not bind directly to DNA but exerts its function by interacting with the cellular proteins RBPJκ (CBF1) and, on the more complex LMP1 promoter, also Spi1 (PU.1), tethered to cognate response elements (12, 17, 20, 45, 46). Transcriptional activation is induced by binding of the C-terminal acidic domain (5) to components of the basal RNA polymerase II transcription machinery, such as RPA70, TAF40, TFIIB, and TFIIH (38, 39), and recruitment of the coactivators p300, CBP, and PCAF histone deacetylase (14, 41). In addition, by attracting the hSWI/SNF complex (42, 43) and targeting histone H1 (9, 34), EBNA2 likely promotes relief of nucleosome-mediated gene repression. We have recently shown that EBNA2 binds to DP103, a novel member of the DEAD-box family of putative RNA helicases (10). DP103 is a ubiquitously expressed 103-kDa phosphoprotein with an RNA-dependent ATPase activity; its other functions, in particular with regard to its interaction with EBNA2, remained unknown. While the work presented here was in progress, an interaction of DP103 (alternatively called Gemin3 [2]) and the survival motor neuron (SMN) protein, and their respective murine homologues, were described in two independent studies (1, 2). SMN is part of a multiprotein complex containing SIP1, DP103 (Gemin3), GIP1 (Gemin4), and several Sm proteins that is involved in the assembly and nuclear regeneration of snRNPs and spliceosomes (2, 7, 25, 31). Both SMN and DP103 are localized in the cytoplasm and distinct nuclear structures, described as coiled bodies and gems (gemini of coiled bodies) (2, 21, 31). Mutations in the SMN gene result in spinal muscular atrophy (SMA), a recessive genetic disease with loss of α-motor neurons in the spinal cord, leading to muscle weakness and subsequent death. The SMN gene exists in two inverted copies within the same chromosomal region on chromosome 5q13 (19). In most SMA patients, a mutated telomeric form of the SMN gene results in a nonfunctional exon 7-deleted SMN, unable to self-associate (22), which cannot be compensated by the low amounts of full-length SMN protein expressed from the centromeric allele (24). In a few cases of SMA, point mutations were described which exchange amino acid (aa) 272 (Y272C) (19) or aa 134 (E134K) (4), affecting a putative RNA binding tudor domain (26). Furthermore, knockout of the murine SMN gene or its yeast homologue Yab8p resulted in a lethal phenotype (11, 28, 35). Interestingly, a role for SMN in transcriptional regulation has been implicated, since SMN was shown to interact with the bovine papillomavirus E2 transactivator and to coactivate an E2-responsive viral promoter (1, 36). Furthermore, Ou et al. demonstrated that murine dp103 is also involved in transcriptional regulation by negatively modulating the expression of steroidogenic factor-1 (27). Finally, the SMN complex has recently been shown to associate with the C-terminal domain (CTD) of RNA polymerase II (30), although the functional consequences of this interaction have not yet been elucidated. Searching for DP103-associated cellular proteins by using the yeast two-hybrid system, we also identified SMN as an interaction partner of DP103. Here, we show that this interaction is also relevant in B cells and that SMN is able to coactivate the viral LMP1 promoter in the presence of EBNA2 in vitro and in vivo. Data obtained from analyzing different EBNA2, DP103, and SMN mutants regarding their binding domains, subcellular distribution, and influence on EBNA2-mediated transactivation suggest that SMN is a novel factor involved in EBNA2-mediated transactivation of the viral LMP1 promoter: by targeting of DP103 within spliceosomal complexes, EBNA2 subsequently releases transcriptionally active SMN, which functions as a coactivator, likely within the RNA polymerase II transcription complex.

International audience; The mRNA targets of microRNAs (miRNAs) can be identified by immunoprecipitation of Argonaute (Ago) protein-containing RNA-induced silencing complexes (RISCs) followed by microarray analysis (RIP-Chip). Here we used Ago2-based RIP-Chip to identify transcripts targeted by Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs (n = 114), Epstein-Barr virus (EBV) miRNAs (n = 44), and cellular miRNAs (n = 2337) in six latently infected or stably transduced human B cell lines. Of the six KSHV miRNA targets chosen for validation, four showed regulation via their 3'UTR, while two showed regulation via binding sites within coding sequences. Two genes governing cellular transport processes (TOMM22 and IPO7) were confirmed to be targeted by EBV miRNAs. A significant number of viral miRNA targets were upregulated in infected cells, suggesting that viral miRNAs preferentially target cellular genes induced upon infection. Transcript half-life both of cellular and viral miRNA targets negatively correlated with recruitment to RISC complexes, indicating that RIP-Chip offers a quantitative estimate of miRNA function.