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Epstein-Barr virus BART microRNAs are produced from a large intron prior to splicing.

ABSTRACT: Latent Epstein-Barr virus (EBV) infection is associated with several lymphoproliferative disorders, including posttransplant lymphoma, Hodgkin's disease, and Burkitt's lymphoma, as well as nasopharyngeal carcinoma (NPC). Twenty-nine microRNAs (miRNAs) have been identified that are transcribed during latent infection from three clusters in the EBV genome. Two of the three clusters of miRNAs are made from the BamHI A rightward transcripts (BARTs), a set of alternatively spliced transcripts that are highly abundant in NPC but have not been shown to produce a detectable protein. This study indicates that while the BART miRNAs are located in the first four introns of the transcripts, processing of the pre-miRNAs from the primary transcript occurs prior to completion of the splicing reaction. Additionally, production of the BART miRNAs correlates with accumulation of a spliced mRNA in which exon 1 is joined directly to exon 3, suggesting that this form of the transcript may favor production of miRNAs. Sequence variations and processing of pre-miRNAs to the mature form also may account for various differences in miRNA abundance. Importantly, residual intronic pieces that result from processing of the pre-miRNAs were detected in the nucleus. The predicted structures of these pieces suggest there is a bias or temporal pattern to the production of the individual pre-miRNAs. These findings indicate that multiple factors contribute to the production of the BART miRNAs and to the apparent differences in abundance between the individual miRNAs of the cluster.

SUBMITTER: Edwards RH

PROVIDER: S-EPMC2546912 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Epstein-Barr virus BART microRNAs are produced from a large intron prior to splicing.

Edwards Rachel Hood RH Marquitz Aron R AR Raab-Traub Nancy N

Journal of virology 20080709 18

Latent Epstein-Barr virus (EBV) infection is associated with several lymphoproliferative disorders, including posttransplant lymphoma, Hodgkin's disease, and Burkitt's lymphoma, as well as nasopharyngeal carcinoma (NPC). Twenty-nine microRNAs (miRNAs) have been identified that are transcribed during latent infection from three clusters in the EBV genome. Two of the three clusters of miRNAs are made from the BamHI A rightward transcripts (BARTs), a set of alternatively spliced transcripts that ar ...[more]

PMID: 18614630

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Project description:UnlabelledEpstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinase inhibitor PS-1145 inhibited NF-κB activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-κB activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-κB signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-κB activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC.ImportanceNasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-κB signaling. EBV LMP1 is a potent activator of NF-κB signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-κB signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-κB signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-κB signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

Project description:This study assessed the effects of Epstein-Barr virus (EBV) and one form of virally encoded BART long noncoding RNAs (lncRNAs) on cellular expression in epithelial cells grown in vitro and as tumors in vivo determined by high-throughput RNA sequencing of mRNA and small RNAs. Hierarchical clustering based on gene expression distinguished the cell lines from the tumors and distinguished the EBV-positive tumors and the BART tumors from the EBV-negative tumors. EBV and BART expression also induced specific expression changes in cellular microRNAs (miRs) and lncRNAs. Multiple known and predicted targets of the viral miRs, the induced cellular miRs, and lncRNAs were identified in the altered gene set. The changes in expression in vivo indicated that the suppression of growth pathways in vivo reflects increased expression of cellular miRs in all tumors. In the EBV and BART tumors, many of the targets of the induced miRs were not changed and the seed sequences of the nonfunctional miRs were found to have homologous regions within the BART lncRNA. The inhibition of these miR effects on known targets suggests that these induced miRs have reduced function due to sponging by the BART lncRNA. This composite analysis identified the effects of EBV on cellular miRs and lncRNAs with a functional readout through identification of the simultaneous effects on gene expression. Major shifts in gene expression in vivo are likely mediated by effects on cellular noncoding RNAs. Additionally, a predicted property of the BART lncRNA is to functionally inhibit the induced cellular miRs. IMPORTANCE This study identified the total effects of EBV and a viral long noncoding RNA (BART lncRNA) on cellular RNA expression when grown as cells in culture and when grown as tumors in immunodeficient mice. The effects on cellular mRNA expression, lncRNA expression, and cellular and viral miR expression were determined using next-generation sequencing (NGS) and bioinformatics functional analysis. Many cellular growth pathways that are activated during growth in culture are decreased during growth as tumors. This study shows that these changes in expression are accompanied by induction of cellular-growth-inhibitory miRs. However, in the EBV tumors and in tumors expressing the BART lncRNA, many of the known targets of the inhibitory miRs are not affected. Regions of strong homology to the seed sequences of these miRs were identified in the BART lncRNA. These findings suggest that the BART lncRNA functions as a sponge for growth-inhibitory miRs.

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Epstein-Barr virus BART microRNAs are produced from a large intron prior to splicing.

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Epstein-Barr virus BART microRNAs are produced from a large intron prior to splicing.

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