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Proteomics and Transcriptomics of BJAB Cells Expressing the Epstein-Barr Virus Noncoding RNAs EBER1 and EBER2.

Pimienta G, Fok V, Haslip M, Nagy M, Takyar S, Steitz JA - PLoS ONE (2015)

Bottom Line: In the mRNA-seq data, the mRNA levels for some of the proteins changing in the SILAC data did not change.The CCL3, CCR7, IL10 and VEGFA proteins promote cell proliferation and are associated with EBV-mediated lymphomas.We previously found that EBER1 interacts with AUF1 in vivo and proposed stabilization of ARE-containing mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
In Epstein-Barr virus (EBV) latent infection, the EBV-encoded RNAs EBER1 and EBER2 accumulate in the host cell nucleus to ~10(6) copies. While the expression of EBERs in cell lines is associated with transformation, a mechanistic explanation of their roles in EBV latency remains elusive. To identify EBER-specific gene expression features, we compared the proteome and mRNA transcriptome from BJAB cells (an EBV-negative B lymphoma cell line) stably transfected with an empty plasmid or with one carrying both EBER genes. We identified ~1800 proteins with at least 2 SILAC pair measurements, of which only 8 and 12 were up- and downregulated ≥ 2-fold, respectively. One upregulated protein was PIK3AP1, a B-cell specific protein adapter known to activate the PI3K-AKT signaling pathway, which regulates alternative splicing and translation in addition to its pro-survival effects. In the mRNA-seq data, the mRNA levels for some of the proteins changing in the SILAC data did not change. We instead observed isoform switch events. We validated the most relevant findings with biochemical assays. These corroborated the upregulation of PIK3AP1 and AKT activation in BJAB cells expressing high levels of both EBERs and EBNA1 (a surrogate of Burkitt's lymphoma EBV latency I) relative to those expressing only EBNA1. The mRNA-seq data in these cells showed multiple upregulated oncogenes whose mRNAs are enriched for 3´-UTR AU-rich elements (AREs), such as ccl3, ccr7, il10, vegfa and zeb1. The CCL3, CCR7, IL10 and VEGFA proteins promote cell proliferation and are associated with EBV-mediated lymphomas. In EBV latency, ZEB1 represses the transcription of ZEBRA, an EBV lytic phase activation factor. We previously found that EBER1 interacts with AUF1 in vivo and proposed stabilization of ARE-containing mRNAs. Thus, the ~10(6) copies of EBER1 may promote not only cell proliferation due to an increase in the levels of ARE-containing genes like ccl3, ccr7, il10, and vegfa, but also the maintenance of latency, through higher levels of zeb1.

No MeSH data available.


Related in: MedlinePlus

GO analysis and tumorigenic signature.Using the DAVID web-based portal (http://david.abcc.ncifcrf.gov/), we performed a GO analysis of the two datasets (low and high EBER1/2 expression) based on the main PANTHER classification terms, Molecular Function (MF) and Biological Property (BP). (A) GO analysis of the BJAB-EBER1/2 vs BJAB-CTL comparison (low EBER1/2 expression levels). Right and left plots indicate the enrichment in MF and BP terms, respectively. (B) As in the previous panel, GO analysis of the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison (high EBER1/2 levels). (C) Figure adapted from Hanahan and Weinberg [37] to highlight the oncogenic signature enriched in BJAB-EBNA1-EBER1/2 cells. (D) qRT-PCR validation assays for il10, vegfa and zeb1 mRNAs from three biological replicates per comparison. The left plot shows an overlay of the BJAB-EBER1/2 vs BJAB-CTL (blue) and BJAB-EBNA1-EBER1-/2 vs BJAB-EBNA1 (red) fold-changes. The right plot shows the BJAB-B1 vs BJAB-parental fold-changes. All values were normalized to ACTB as a control. (E) WB assays with an antibody specific for ZEB1 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Densitometry values and error bars reflect three independent biological replicates.
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pone.0124638.g005: GO analysis and tumorigenic signature.Using the DAVID web-based portal (http://david.abcc.ncifcrf.gov/), we performed a GO analysis of the two datasets (low and high EBER1/2 expression) based on the main PANTHER classification terms, Molecular Function (MF) and Biological Property (BP). (A) GO analysis of the BJAB-EBER1/2 vs BJAB-CTL comparison (low EBER1/2 expression levels). Right and left plots indicate the enrichment in MF and BP terms, respectively. (B) As in the previous panel, GO analysis of the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison (high EBER1/2 levels). (C) Figure adapted from Hanahan and Weinberg [37] to highlight the oncogenic signature enriched in BJAB-EBNA1-EBER1/2 cells. (D) qRT-PCR validation assays for il10, vegfa and zeb1 mRNAs from three biological replicates per comparison. The left plot shows an overlay of the BJAB-EBER1/2 vs BJAB-CTL (blue) and BJAB-EBNA1-EBER1-/2 vs BJAB-EBNA1 (red) fold-changes. The right plot shows the BJAB-B1 vs BJAB-parental fold-changes. All values were normalized to ACTB as a control. (E) WB assays with an antibody specific for ZEB1 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Densitometry values and error bars reflect three independent biological replicates.

Mentions: To more thoroughly investigate the effects of high compared to low EBER expression, we interpreted the mRNA-seq data from BJAB-EBNA1-EBER1/2 and BJAB-EBNA1 cells. In the first replicate, Cuffdiff computed a total of 14,345 genes, of which 221 genes were upregulated (1.5%) and 506 downregulated (3.5%) significantly (S8 File). Compared to the 1.5% of upregulated genes in the first replicate of the BJAB-EBER1/2 vs BJAB-CTL comparison, the number of genes with a significant upregulation in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 dataset was similar even though their identity was not entirely the same (S9 File). A GO analysis of the genes changing significantly in total mRNA levels (according to their q-value) in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison, revealed a tumorigenic gene expression signature (Fig 5A), absent in the data from BJAB cells that express low EBER levels (Fig 5B).


Proteomics and Transcriptomics of BJAB Cells Expressing the Epstein-Barr Virus Noncoding RNAs EBER1 and EBER2.

Pimienta G, Fok V, Haslip M, Nagy M, Takyar S, Steitz JA - PLoS ONE (2015)

GO analysis and tumorigenic signature.Using the DAVID web-based portal (http://david.abcc.ncifcrf.gov/), we performed a GO analysis of the two datasets (low and high EBER1/2 expression) based on the main PANTHER classification terms, Molecular Function (MF) and Biological Property (BP). (A) GO analysis of the BJAB-EBER1/2 vs BJAB-CTL comparison (low EBER1/2 expression levels). Right and left plots indicate the enrichment in MF and BP terms, respectively. (B) As in the previous panel, GO analysis of the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison (high EBER1/2 levels). (C) Figure adapted from Hanahan and Weinberg [37] to highlight the oncogenic signature enriched in BJAB-EBNA1-EBER1/2 cells. (D) qRT-PCR validation assays for il10, vegfa and zeb1 mRNAs from three biological replicates per comparison. The left plot shows an overlay of the BJAB-EBER1/2 vs BJAB-CTL (blue) and BJAB-EBNA1-EBER1-/2 vs BJAB-EBNA1 (red) fold-changes. The right plot shows the BJAB-B1 vs BJAB-parental fold-changes. All values were normalized to ACTB as a control. (E) WB assays with an antibody specific for ZEB1 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Densitometry values and error bars reflect three independent biological replicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487896&req=5

pone.0124638.g005: GO analysis and tumorigenic signature.Using the DAVID web-based portal (http://david.abcc.ncifcrf.gov/), we performed a GO analysis of the two datasets (low and high EBER1/2 expression) based on the main PANTHER classification terms, Molecular Function (MF) and Biological Property (BP). (A) GO analysis of the BJAB-EBER1/2 vs BJAB-CTL comparison (low EBER1/2 expression levels). Right and left plots indicate the enrichment in MF and BP terms, respectively. (B) As in the previous panel, GO analysis of the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison (high EBER1/2 levels). (C) Figure adapted from Hanahan and Weinberg [37] to highlight the oncogenic signature enriched in BJAB-EBNA1-EBER1/2 cells. (D) qRT-PCR validation assays for il10, vegfa and zeb1 mRNAs from three biological replicates per comparison. The left plot shows an overlay of the BJAB-EBER1/2 vs BJAB-CTL (blue) and BJAB-EBNA1-EBER1-/2 vs BJAB-EBNA1 (red) fold-changes. The right plot shows the BJAB-B1 vs BJAB-parental fold-changes. All values were normalized to ACTB as a control. (E) WB assays with an antibody specific for ZEB1 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Densitometry values and error bars reflect three independent biological replicates.
Mentions: To more thoroughly investigate the effects of high compared to low EBER expression, we interpreted the mRNA-seq data from BJAB-EBNA1-EBER1/2 and BJAB-EBNA1 cells. In the first replicate, Cuffdiff computed a total of 14,345 genes, of which 221 genes were upregulated (1.5%) and 506 downregulated (3.5%) significantly (S8 File). Compared to the 1.5% of upregulated genes in the first replicate of the BJAB-EBER1/2 vs BJAB-CTL comparison, the number of genes with a significant upregulation in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 dataset was similar even though their identity was not entirely the same (S9 File). A GO analysis of the genes changing significantly in total mRNA levels (according to their q-value) in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison, revealed a tumorigenic gene expression signature (Fig 5A), absent in the data from BJAB cells that express low EBER levels (Fig 5B).

Bottom Line: In the mRNA-seq data, the mRNA levels for some of the proteins changing in the SILAC data did not change.The CCL3, CCR7, IL10 and VEGFA proteins promote cell proliferation and are associated with EBV-mediated lymphomas.We previously found that EBER1 interacts with AUF1 in vivo and proposed stabilization of ARE-containing mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
In Epstein-Barr virus (EBV) latent infection, the EBV-encoded RNAs EBER1 and EBER2 accumulate in the host cell nucleus to ~10(6) copies. While the expression of EBERs in cell lines is associated with transformation, a mechanistic explanation of their roles in EBV latency remains elusive. To identify EBER-specific gene expression features, we compared the proteome and mRNA transcriptome from BJAB cells (an EBV-negative B lymphoma cell line) stably transfected with an empty plasmid or with one carrying both EBER genes. We identified ~1800 proteins with at least 2 SILAC pair measurements, of which only 8 and 12 were up- and downregulated ≥ 2-fold, respectively. One upregulated protein was PIK3AP1, a B-cell specific protein adapter known to activate the PI3K-AKT signaling pathway, which regulates alternative splicing and translation in addition to its pro-survival effects. In the mRNA-seq data, the mRNA levels for some of the proteins changing in the SILAC data did not change. We instead observed isoform switch events. We validated the most relevant findings with biochemical assays. These corroborated the upregulation of PIK3AP1 and AKT activation in BJAB cells expressing high levels of both EBERs and EBNA1 (a surrogate of Burkitt's lymphoma EBV latency I) relative to those expressing only EBNA1. The mRNA-seq data in these cells showed multiple upregulated oncogenes whose mRNAs are enriched for 3´-UTR AU-rich elements (AREs), such as ccl3, ccr7, il10, vegfa and zeb1. The CCL3, CCR7, IL10 and VEGFA proteins promote cell proliferation and are associated with EBV-mediated lymphomas. In EBV latency, ZEB1 represses the transcription of ZEBRA, an EBV lytic phase activation factor. We previously found that EBER1 interacts with AUF1 in vivo and proposed stabilization of ARE-containing mRNAs. Thus, the ~10(6) copies of EBER1 may promote not only cell proliferation due to an increase in the levels of ARE-containing genes like ccl3, ccr7, il10, and vegfa, but also the maintenance of latency, through higher levels of zeb1.

No MeSH data available.


Related in: MedlinePlus