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An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival.

Choy EY, Siu KL, Kok KH, Lung RW, Tsang CM, To KF, Kwong DL, Tsao SW, Jin DY - J. Exp. Med. (2008)

Bottom Line: In addition, PUMA was found to be significantly underexpressed in approximately 60% of human NPC tissues.Although expression of miR-BART5 rendered NPC and EBV-GC cells less sensitive to proapoptotic agents, apoptosis can be triggered by depleting miR-BART5 or inducing the expression of PUMA.Collectively, our findings suggest that EBV encodes an miRNA to facilitate the establishment of latent infection by promoting host cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong.

ABSTRACT
Epstein-Barr virus (EBV) is a herpesvirus associated with nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and other malignancies. EBV is the first human virus found to express microRNAs (miRNAs), the functions of which remain largely unknown. We report on the regulation of a cellular protein named p53 up-regulated modulator of apoptosis (PUMA) by an EBV miRNA known as miR-BART5, which is abundantly expressed in NPC and EBV-GC cells. Modulation of PUMA expression by miR-BART5 and anti-miR-BART5 oligonucleotide was demonstrated in EBV-positive cells. In addition, PUMA was found to be significantly underexpressed in approximately 60% of human NPC tissues. Although expression of miR-BART5 rendered NPC and EBV-GC cells less sensitive to proapoptotic agents, apoptosis can be triggered by depleting miR-BART5 or inducing the expression of PUMA. Collectively, our findings suggest that EBV encodes an miRNA to facilitate the establishment of latent infection by promoting host cell survival.

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PUMA is a cellular target of miR-BART5. (A) Target site of miR-BART5 in 3′ UTR of PUMA. The top shows the sequences in the 3′ UTR of PUMA that base pair with miR-BART5. The bottom shows the conservation of the target site sequences in different species of vertebrates. Conservative nucleotides are boxed. (B) Functional characterization of miR-BART5 target site. pGL3-PUMA.UTR has target sites of miR-BART5 inserted into the 3′ UTR of luciferase gene. This reporter construct was cotransfected into HeLa cells with an expression vector for either miR-BART4 or miR-BART5. Plasmids pGL3-BART5.UTR and pGL3-GAPDH served as positive and negative controls, respectively. These control plasmids are different from pGL3-PUMA.UTR only in the 3′ UTR of luciferase gene. pGL3-BART5.UTR contains target sequences perfectly matched with miR-BART5, whereas pGL3-GAPDH.UTR harbors irrelevant GAPDH sequences. In pGL3-mutPUMA.UTR, the target sites of miR-BART5 in the PUMA 3′ UTR were mutated as indicated in the top. The relative luciferase activity was obtained by normalizing the readings of the firefly luciferase (Fluc) activity from the pGL3 vector with those of the Renilla reniformis luciferase activity from pRL-CMV. Results represent mean ± SD from three independent experiments. (C and D) Functionality of miR-BART5 target site in PUMA 3′ UTR. A single copy of the entire PUMA 3′ UTR sequence was inserted into the 3′ UTR of luciferase gene to generate pGL3-PUMA.FL.UTR. This reporter construct was cotransfected into HeLa cells with an expression vector for either miR-BART4 or miR-BART5, or with commercially available synthetic RNA oligonucleotides pre–miRNA-NC or pre–miR-BART5. Plasmid pGL3-PUMA.FL.rUTR, in which a reversed single copy of the PUMA 3′ UTR sequence was inserted into the 3′ UTR of luciferase gene, served as a negative control. Error bars indicate SD (n = 3). *, P = 0.2819 (by Student's t test); **, P = 0.0346; #, P = 0.0042. ^, P = 0.0069.
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fig1: PUMA is a cellular target of miR-BART5. (A) Target site of miR-BART5 in 3′ UTR of PUMA. The top shows the sequences in the 3′ UTR of PUMA that base pair with miR-BART5. The bottom shows the conservation of the target site sequences in different species of vertebrates. Conservative nucleotides are boxed. (B) Functional characterization of miR-BART5 target site. pGL3-PUMA.UTR has target sites of miR-BART5 inserted into the 3′ UTR of luciferase gene. This reporter construct was cotransfected into HeLa cells with an expression vector for either miR-BART4 or miR-BART5. Plasmids pGL3-BART5.UTR and pGL3-GAPDH served as positive and negative controls, respectively. These control plasmids are different from pGL3-PUMA.UTR only in the 3′ UTR of luciferase gene. pGL3-BART5.UTR contains target sequences perfectly matched with miR-BART5, whereas pGL3-GAPDH.UTR harbors irrelevant GAPDH sequences. In pGL3-mutPUMA.UTR, the target sites of miR-BART5 in the PUMA 3′ UTR were mutated as indicated in the top. The relative luciferase activity was obtained by normalizing the readings of the firefly luciferase (Fluc) activity from the pGL3 vector with those of the Renilla reniformis luciferase activity from pRL-CMV. Results represent mean ± SD from three independent experiments. (C and D) Functionality of miR-BART5 target site in PUMA 3′ UTR. A single copy of the entire PUMA 3′ UTR sequence was inserted into the 3′ UTR of luciferase gene to generate pGL3-PUMA.FL.UTR. This reporter construct was cotransfected into HeLa cells with an expression vector for either miR-BART4 or miR-BART5, or with commercially available synthetic RNA oligonucleotides pre–miRNA-NC or pre–miR-BART5. Plasmid pGL3-PUMA.FL.rUTR, in which a reversed single copy of the PUMA 3′ UTR sequence was inserted into the 3′ UTR of luciferase gene, served as a negative control. Error bars indicate SD (n = 3). *, P = 0.2819 (by Student's t test); **, P = 0.0346; #, P = 0.0042. ^, P = 0.0069.

Mentions: The predicted miR-BART5 target site in the 3′ UTR of PUMA exhibited good complementarity to miR-BART5 and was highly conserved among human, chimp, rhesus monkey, and mouse (Fig. 1 A). Although a 7-nt stretch that perfectly matches the 5′ seed region of miR-BART5 was found in the predicted site, there were multiple mismatches in the central region (Fig. 1 A, top).


An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival.

Choy EY, Siu KL, Kok KH, Lung RW, Tsang CM, To KF, Kwong DL, Tsao SW, Jin DY - J. Exp. Med. (2008)

PUMA is a cellular target of miR-BART5. (A) Target site of miR-BART5 in 3′ UTR of PUMA. The top shows the sequences in the 3′ UTR of PUMA that base pair with miR-BART5. The bottom shows the conservation of the target site sequences in different species of vertebrates. Conservative nucleotides are boxed. (B) Functional characterization of miR-BART5 target site. pGL3-PUMA.UTR has target sites of miR-BART5 inserted into the 3′ UTR of luciferase gene. This reporter construct was cotransfected into HeLa cells with an expression vector for either miR-BART4 or miR-BART5. Plasmids pGL3-BART5.UTR and pGL3-GAPDH served as positive and negative controls, respectively. These control plasmids are different from pGL3-PUMA.UTR only in the 3′ UTR of luciferase gene. pGL3-BART5.UTR contains target sequences perfectly matched with miR-BART5, whereas pGL3-GAPDH.UTR harbors irrelevant GAPDH sequences. In pGL3-mutPUMA.UTR, the target sites of miR-BART5 in the PUMA 3′ UTR were mutated as indicated in the top. The relative luciferase activity was obtained by normalizing the readings of the firefly luciferase (Fluc) activity from the pGL3 vector with those of the Renilla reniformis luciferase activity from pRL-CMV. Results represent mean ± SD from three independent experiments. (C and D) Functionality of miR-BART5 target site in PUMA 3′ UTR. A single copy of the entire PUMA 3′ UTR sequence was inserted into the 3′ UTR of luciferase gene to generate pGL3-PUMA.FL.UTR. This reporter construct was cotransfected into HeLa cells with an expression vector for either miR-BART4 or miR-BART5, or with commercially available synthetic RNA oligonucleotides pre–miRNA-NC or pre–miR-BART5. Plasmid pGL3-PUMA.FL.rUTR, in which a reversed single copy of the PUMA 3′ UTR sequence was inserted into the 3′ UTR of luciferase gene, served as a negative control. Error bars indicate SD (n = 3). *, P = 0.2819 (by Student's t test); **, P = 0.0346; #, P = 0.0042. ^, P = 0.0069.
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Related In: Results  -  Collection

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fig1: PUMA is a cellular target of miR-BART5. (A) Target site of miR-BART5 in 3′ UTR of PUMA. The top shows the sequences in the 3′ UTR of PUMA that base pair with miR-BART5. The bottom shows the conservation of the target site sequences in different species of vertebrates. Conservative nucleotides are boxed. (B) Functional characterization of miR-BART5 target site. pGL3-PUMA.UTR has target sites of miR-BART5 inserted into the 3′ UTR of luciferase gene. This reporter construct was cotransfected into HeLa cells with an expression vector for either miR-BART4 or miR-BART5. Plasmids pGL3-BART5.UTR and pGL3-GAPDH served as positive and negative controls, respectively. These control plasmids are different from pGL3-PUMA.UTR only in the 3′ UTR of luciferase gene. pGL3-BART5.UTR contains target sequences perfectly matched with miR-BART5, whereas pGL3-GAPDH.UTR harbors irrelevant GAPDH sequences. In pGL3-mutPUMA.UTR, the target sites of miR-BART5 in the PUMA 3′ UTR were mutated as indicated in the top. The relative luciferase activity was obtained by normalizing the readings of the firefly luciferase (Fluc) activity from the pGL3 vector with those of the Renilla reniformis luciferase activity from pRL-CMV. Results represent mean ± SD from three independent experiments. (C and D) Functionality of miR-BART5 target site in PUMA 3′ UTR. A single copy of the entire PUMA 3′ UTR sequence was inserted into the 3′ UTR of luciferase gene to generate pGL3-PUMA.FL.UTR. This reporter construct was cotransfected into HeLa cells with an expression vector for either miR-BART4 or miR-BART5, or with commercially available synthetic RNA oligonucleotides pre–miRNA-NC or pre–miR-BART5. Plasmid pGL3-PUMA.FL.rUTR, in which a reversed single copy of the PUMA 3′ UTR sequence was inserted into the 3′ UTR of luciferase gene, served as a negative control. Error bars indicate SD (n = 3). *, P = 0.2819 (by Student's t test); **, P = 0.0346; #, P = 0.0042. ^, P = 0.0069.
Mentions: The predicted miR-BART5 target site in the 3′ UTR of PUMA exhibited good complementarity to miR-BART5 and was highly conserved among human, chimp, rhesus monkey, and mouse (Fig. 1 A). Although a 7-nt stretch that perfectly matches the 5′ seed region of miR-BART5 was found in the predicted site, there were multiple mismatches in the central region (Fig. 1 A, top).

Bottom Line: In addition, PUMA was found to be significantly underexpressed in approximately 60% of human NPC tissues.Although expression of miR-BART5 rendered NPC and EBV-GC cells less sensitive to proapoptotic agents, apoptosis can be triggered by depleting miR-BART5 or inducing the expression of PUMA.Collectively, our findings suggest that EBV encodes an miRNA to facilitate the establishment of latent infection by promoting host cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong.

ABSTRACT
Epstein-Barr virus (EBV) is a herpesvirus associated with nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and other malignancies. EBV is the first human virus found to express microRNAs (miRNAs), the functions of which remain largely unknown. We report on the regulation of a cellular protein named p53 up-regulated modulator of apoptosis (PUMA) by an EBV miRNA known as miR-BART5, which is abundantly expressed in NPC and EBV-GC cells. Modulation of PUMA expression by miR-BART5 and anti-miR-BART5 oligonucleotide was demonstrated in EBV-positive cells. In addition, PUMA was found to be significantly underexpressed in approximately 60% of human NPC tissues. Although expression of miR-BART5 rendered NPC and EBV-GC cells less sensitive to proapoptotic agents, apoptosis can be triggered by depleting miR-BART5 or inducing the expression of PUMA. Collectively, our findings suggest that EBV encodes an miRNA to facilitate the establishment of latent infection by promoting host cell survival.

Show MeSH
Related in: MedlinePlus