Limits...
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

SILAC ratios of the proteins identified.Log2 SILAC ratios (BJAB-EBER1/2 vs BJAB-CTL) plotted against the number of proteins identified. Ratios with ≥ 1 SILAC pair count are colored blue and those with ≥ 2 counts red. Representative proteins upregulated are indicated.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4487896&req=5

pone.0124638.g002: SILAC ratios of the proteins identified.Log2 SILAC ratios (BJAB-EBER1/2 vs BJAB-CTL) plotted against the number of proteins identified. Ratios with ≥ 1 SILAC pair count are colored blue and those with ≥ 2 counts red. Representative proteins upregulated are indicated.

Mentions: Changes in the levels of most proteins were subtle (Fig 2). In the list of individual proteins with at least 2 SILAC counts, we found 8 upregulated and 12 downregulated ≥ 2-fold (Table 1). All of the up- and downregulated proteins had good posterior error probability (PEP) scores (S2 File). We also confirmed that their tandem mass spectrometry (MS/MS) fragmentation spectra were of good quality–the spectra had a good signal-to-noise ratio and most amino acids in the peptide sequence were explained by the MS/MS data (S1B, S1C and S1D Fig).


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)

SILAC ratios of the proteins identified.Log2 SILAC ratios (BJAB-EBER1/2 vs BJAB-CTL) plotted against the number of proteins identified. Ratios with ≥ 1 SILAC pair count are colored blue and those with ≥ 2 counts red. Representative proteins upregulated are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124638.g002: SILAC ratios of the proteins identified.Log2 SILAC ratios (BJAB-EBER1/2 vs BJAB-CTL) plotted against the number of proteins identified. Ratios with ≥ 1 SILAC pair count are colored blue and those with ≥ 2 counts red. Representative proteins upregulated are indicated.
Mentions: Changes in the levels of most proteins were subtle (Fig 2). In the list of individual proteins with at least 2 SILAC counts, we found 8 upregulated and 12 downregulated ≥ 2-fold (Table 1). All of the up- and downregulated proteins had good posterior error probability (PEP) scores (S2 File). We also confirmed that their tandem mass spectrometry (MS/MS) fragmentation spectra were of good quality–the spectra had a good signal-to-noise ratio and most amino acids in the peptide sequence were explained by the MS/MS data (S1B, S1C and S1D Fig).

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