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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

Validation by qRT-PCR of zeb1 alternative isoform abundances in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison.(A) The cartoon shows the mRNA-seq paired-end reads aligned by TopHat to the zeb1 gene and indicates the alternative isoforms identified by Cufflinks in the bioinformatics analysis. The isoforms with a significant fold-change in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison are colored blue. (B) Isoform abundances calculated by Cufflinks for each alternative isoform identified. The isoforms calculated to have changed significantly by Cufflinks are colored blue. (C) Validation by qRT-PCR of the indicated isoforms. The qRT-PCR values were normalized to 18S rRNA.
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pone.0124638.g006: Validation by qRT-PCR of zeb1 alternative isoform abundances in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison.(A) The cartoon shows the mRNA-seq paired-end reads aligned by TopHat to the zeb1 gene and indicates the alternative isoforms identified by Cufflinks in the bioinformatics analysis. The isoforms with a significant fold-change in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison are colored blue. (B) Isoform abundances calculated by Cufflinks for each alternative isoform identified. The isoforms calculated to have changed significantly by Cufflinks are colored blue. (C) Validation by qRT-PCR of the indicated isoforms. The qRT-PCR values were normalized to 18S rRNA.

Mentions: According to Cufflinks, the changes in zeb1 individual isoform abundances were mainly due to alternative promoter-usage (Fig 6A and S10 and S12 Files). To demonstrate the accuracy of Cufflinks, we validated the changes in isoform abundance calculated for the gene zeb1 (Fig 6A, 6B and 6C). We also validated the gene vegfa at the isoform level (Fig 7A, 7B and 7C). The vegfa gene has 19 alternative isoforms annotated in RefSeq and multiple alternative translation initiation sites [47] (Fig 7A). The complexity of this gene makes the analysis difficult for Cufflinks because the statistical power of this and other transcript-abundance computational tools decreases for genes with more than three isoforms [48]. Our mRNA-seq data indicated that only four of the 19 RefSeq-annotated isoforms were expressed at detectable levels, all upregulated with different fold-changes in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison (Fig 7B). We designed specific primers for three of the four isoforms upregulated in the mRNA-seq data and were able to validate these by qRT-PCR assays (Fig 7C).


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)

Validation by qRT-PCR of zeb1 alternative isoform abundances in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison.(A) The cartoon shows the mRNA-seq paired-end reads aligned by TopHat to the zeb1 gene and indicates the alternative isoforms identified by Cufflinks in the bioinformatics analysis. The isoforms with a significant fold-change in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison are colored blue. (B) Isoform abundances calculated by Cufflinks for each alternative isoform identified. The isoforms calculated to have changed significantly by Cufflinks are colored blue. (C) Validation by qRT-PCR of the indicated isoforms. The qRT-PCR values were normalized to 18S rRNA.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124638.g006: Validation by qRT-PCR of zeb1 alternative isoform abundances in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison.(A) The cartoon shows the mRNA-seq paired-end reads aligned by TopHat to the zeb1 gene and indicates the alternative isoforms identified by Cufflinks in the bioinformatics analysis. The isoforms with a significant fold-change in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison are colored blue. (B) Isoform abundances calculated by Cufflinks for each alternative isoform identified. The isoforms calculated to have changed significantly by Cufflinks are colored blue. (C) Validation by qRT-PCR of the indicated isoforms. The qRT-PCR values were normalized to 18S rRNA.
Mentions: According to Cufflinks, the changes in zeb1 individual isoform abundances were mainly due to alternative promoter-usage (Fig 6A and S10 and S12 Files). To demonstrate the accuracy of Cufflinks, we validated the changes in isoform abundance calculated for the gene zeb1 (Fig 6A, 6B and 6C). We also validated the gene vegfa at the isoform level (Fig 7A, 7B and 7C). The vegfa gene has 19 alternative isoforms annotated in RefSeq and multiple alternative translation initiation sites [47] (Fig 7A). The complexity of this gene makes the analysis difficult for Cufflinks because the statistical power of this and other transcript-abundance computational tools decreases for genes with more than three isoforms [48]. Our mRNA-seq data indicated that only four of the 19 RefSeq-annotated isoforms were expressed at detectable levels, all upregulated with different fold-changes in the BJAB-EBNA1-EBER1/2 vs BJAB-EBNA1 comparison (Fig 7B). We designed specific primers for three of the four isoforms upregulated in the mRNA-seq data and were able to validate these by qRT-PCR assays (Fig 7C).

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