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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

WB and qRT-PCR validation assays.(A) Representative precursor ion SILAC pair MS spectra and WB assay for ADAD2 in the BJAB-EBER1/2 vs BJAB-CTL comparison. (B) WB assays showing the measured fold-changes for La and L22 in the BJAB-EBER1/2 vs BJAB-CTL comparison, which in our SILAC data do not change. (C) Representative precursor ion SILAC pair MS spectra for EIF2B4, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (D) Representative precursor ion SILAC pair MS spectra and WB assay for the FCRLA/1 protein group in the BJAB-EBER1/2 vs BJAB-CTL comparison. Total mRNA level fold-changes based on qRT-PCR assays for the family of FCRLs expressed in BJAB cells in the two indicated comparisons. (E) Representative precursor ion SILAC pair MS spectra for PIK3AP1, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (F) WB assays for total AKT and pAKT in the three indicated comparisons. In all panels, the WB densitometry and qRT-PCR bar plots are based on three independent biological replicates. Error bars indicate the standard deviation. In the qRT-PCR measurements, the values obtained were normalized to ACTB as a control. (G) WB assays using antibodies for ADAD2 and EIF2B4 in total cell lysates from 293T cells transiently transfected (24 hours) with a control plasmid (empty FRT) or one encoding the EBERs (FRT-EBER1/2). Also shown are the WB assays for PIK3AP1, ADAD2 and EIF2B4 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Error bars reflect the standard deviation of three biological replicates.
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pone.0124638.g004: WB and qRT-PCR validation assays.(A) Representative precursor ion SILAC pair MS spectra and WB assay for ADAD2 in the BJAB-EBER1/2 vs BJAB-CTL comparison. (B) WB assays showing the measured fold-changes for La and L22 in the BJAB-EBER1/2 vs BJAB-CTL comparison, which in our SILAC data do not change. (C) Representative precursor ion SILAC pair MS spectra for EIF2B4, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (D) Representative precursor ion SILAC pair MS spectra and WB assay for the FCRLA/1 protein group in the BJAB-EBER1/2 vs BJAB-CTL comparison. Total mRNA level fold-changes based on qRT-PCR assays for the family of FCRLs expressed in BJAB cells in the two indicated comparisons. (E) Representative precursor ion SILAC pair MS spectra for PIK3AP1, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (F) WB assays for total AKT and pAKT in the three indicated comparisons. In all panels, the WB densitometry and qRT-PCR bar plots are based on three independent biological replicates. Error bars indicate the standard deviation. In the qRT-PCR measurements, the values obtained were normalized to ACTB as a control. (G) WB assays using antibodies for ADAD2 and EIF2B4 in total cell lysates from 293T cells transiently transfected (24 hours) with a control plasmid (empty FRT) or one encoding the EBERs (FRT-EBER1/2). Also shown are the WB assays for PIK3AP1, ADAD2 and EIF2B4 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Error bars reflect the standard deviation of three biological replicates.

Mentions: An unbiased GO analysis of the proteomics data cannot be done with the small number of proteins [24] exhibiting a significant fold-change in the SILAC experiment; typically at least 100 gene identification names are needed. Instead, inspection of the literature and GO databases determined that of the 8 upregulated proteins 6 (ADAD2, EIF2B4, FCRLA, FCRL1, PIK3AP1 and UBE2C) are associated with lymphocyte-specific gene expression or oncogenic signaling cascades. For the 12 downregulated proteins, no consensus in known biological functions emerged. From the 6 upregulated proteins mentioned above, we validated 5 (ADAD2, EIF2B4, FCRLA, FCRL1 and PIK3AP1) at the protein and/or mRNA levels by western blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) assays, respectively, in control and EBER1/2-expressing BJAB cells (Fig 4).


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)

WB and qRT-PCR validation assays.(A) Representative precursor ion SILAC pair MS spectra and WB assay for ADAD2 in the BJAB-EBER1/2 vs BJAB-CTL comparison. (B) WB assays showing the measured fold-changes for La and L22 in the BJAB-EBER1/2 vs BJAB-CTL comparison, which in our SILAC data do not change. (C) Representative precursor ion SILAC pair MS spectra for EIF2B4, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (D) Representative precursor ion SILAC pair MS spectra and WB assay for the FCRLA/1 protein group in the BJAB-EBER1/2 vs BJAB-CTL comparison. Total mRNA level fold-changes based on qRT-PCR assays for the family of FCRLs expressed in BJAB cells in the two indicated comparisons. (E) Representative precursor ion SILAC pair MS spectra for PIK3AP1, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (F) WB assays for total AKT and pAKT in the three indicated comparisons. In all panels, the WB densitometry and qRT-PCR bar plots are based on three independent biological replicates. Error bars indicate the standard deviation. In the qRT-PCR measurements, the values obtained were normalized to ACTB as a control. (G) WB assays using antibodies for ADAD2 and EIF2B4 in total cell lysates from 293T cells transiently transfected (24 hours) with a control plasmid (empty FRT) or one encoding the EBERs (FRT-EBER1/2). Also shown are the WB assays for PIK3AP1, ADAD2 and EIF2B4 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Error bars reflect the standard deviation of three biological replicates.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124638.g004: WB and qRT-PCR validation assays.(A) Representative precursor ion SILAC pair MS spectra and WB assay for ADAD2 in the BJAB-EBER1/2 vs BJAB-CTL comparison. (B) WB assays showing the measured fold-changes for La and L22 in the BJAB-EBER1/2 vs BJAB-CTL comparison, which in our SILAC data do not change. (C) Representative precursor ion SILAC pair MS spectra for EIF2B4, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (D) Representative precursor ion SILAC pair MS spectra and WB assay for the FCRLA/1 protein group in the BJAB-EBER1/2 vs BJAB-CTL comparison. Total mRNA level fold-changes based on qRT-PCR assays for the family of FCRLs expressed in BJAB cells in the two indicated comparisons. (E) Representative precursor ion SILAC pair MS spectra for PIK3AP1, plus the corresponding WB and qRT-PCR assays in the BJAB-EBER1/2 vs BJAB-CTL comparison. (F) WB assays for total AKT and pAKT in the three indicated comparisons. In all panels, the WB densitometry and qRT-PCR bar plots are based on three independent biological replicates. Error bars indicate the standard deviation. In the qRT-PCR measurements, the values obtained were normalized to ACTB as a control. (G) WB assays using antibodies for ADAD2 and EIF2B4 in total cell lysates from 293T cells transiently transfected (24 hours) with a control plasmid (empty FRT) or one encoding the EBERs (FRT-EBER1/2). Also shown are the WB assays for PIK3AP1, ADAD2 and EIF2B4 in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison. Error bars reflect the standard deviation of three biological replicates.
Mentions: An unbiased GO analysis of the proteomics data cannot be done with the small number of proteins [24] exhibiting a significant fold-change in the SILAC experiment; typically at least 100 gene identification names are needed. Instead, inspection of the literature and GO databases determined that of the 8 upregulated proteins 6 (ADAD2, EIF2B4, FCRLA, FCRL1, PIK3AP1 and UBE2C) are associated with lymphocyte-specific gene expression or oncogenic signaling cascades. For the 12 downregulated proteins, no consensus in known biological functions emerged. From the 6 upregulated proteins mentioned above, we validated 5 (ADAD2, EIF2B4, FCRLA, FCRL1 and PIK3AP1) at the protein and/or mRNA levels by western blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) assays, respectively, in control and EBER1/2-expressing BJAB cells (Fig 4).

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