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

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1 An Epstein-Barr virus-encoded microrna targets to promote host cell survival The Journal of Experimental Medicine 205(11): , Elizabeth Yee-Wai Choy, Kam-Leung Siu, Kin-Hang Kok, Raymond Wai-Ming Lung, Chi Man Tsang, Ka-Fai To, Dora Lai-Wan Kwong, Sai Wah Tsao, and Dong-Yan Jin Student number: B94B02020 Speaker: Chien-Sin Chen Advisor: Li-Kwan Chang, Ph.D.

2 Epstein-Barr virus (EBV) 2 Belongs to dsdna γ-herpesvirus Capsid diameter: approx. 100 nm Host: Epithelial cells B lymphocytes Chemical inducers (TPA, Na butyrate), Stress Life cycle: Latent/lytic 2 stage Latent phase Latent protein, mirna Pathogenicity: 90% people was infected Hodgkin s disease, Burkitt s lymphoma, and nasopharyngeal carcinoma (NPC) Tumor Lytic phase

3 microrna (mirna) 3 mir-bart5 Small regulatory RNAs ~22 nt in length mirna gene or intronic mirna Target 3 UTR sequence Most target Imperfectly Both cellular and viral mirna are found mir-bart5 mirna 5 UTR Gene 3 UTR mrna

4 Now-known microrna of EBV 4 mir-bart5 Latent genes Lytic genes Previous known mirna Newly identified mirna Latent promoters Lytic promoters Intronic segments Exonic segments EBER: EBV-encoded small RNAs BART: Bam HI-A region rightward transcript

5 p53 up-regulated modulator of apoptosis () 5 Identified in 2001 Bcl-2 family, BH3 only protein -α, -β are 25, 20 kda respectively As a principal mediator of p53 apoptotic signal p53 dependent and independent pathway are identified p53 apoptosis Other than p53, is another hinge of apoptosis apoptosis

6 The story is 6 EBV mir-bart5 immortalization

7 Specific aim 7 Normally Cell death signal, Stress, p53 DNA mrna Cell death EBV infection Latency mirna p53 DNA mrna? Cell death signal, Stress,? Cell survival mir-bart5

8 8 Framework In silica prediction, confirming EBV infected NPC mir-bart5 Cell survival

9 Luciferase reporter assay 9 Luciferase gene 3 UTR mir-bart5 Complementary sequence in 3 UTR (binding site) 3 UTR GAPDH 4 binding sites Binding site mutant Perfectly match with mir-bart5 - + Luciferase gene inhibition

10 mir-bart5 binds 3 UTR of 10 inhib bition - mutant + Luciferase gene

11 is cellular target of mir-bart5 11 plasmid inhibition Luciferase gene Full length 3 UTR Full length 3 UTR Chemical synthetic mirna precursor Luciferase gene Inverted 3 UTR Anti-sense of 3 UTR Intact 3 UTR

12 12 Framework In silica prediction, confirming EBV infected NPC mir-bart5 Cell survival

13 13 Assumption EBV+ EBVinfec ction

14 mir-bart5 was expressed in EBV infected NPC cells 14 EBV NPC HK1 Detect directly (northern) BART5- EBV- C666-1 EBV+ BART5+ EBV-HK1 BART5+ - +

15 was down-regulated in these EBV infected NPC cells 15 EBV - + EBV+ EBV- HK1: EBV- NPC cell line EBV+ B N: noncancerous tissue (normal tissue); T: tumorous tissue (EBV infected NPC cells)

16 16 exogenous BART5 EBV+ Anti-BART5 EBVinfec ction

17 mir-bart5 negatively regulates Endogenous mir-bart expression Endogenous mir-bart HeLa and HK1 are EBV- cell lines C666-1 is EBV+ cell line BART5 Anti-BART5 Exogenous mir-bart5 suppresses -β expression NC: negative control Exogenous anti-mir-bart5 recovers -β and -α expression

18 18 Framework In silica prediction, confirming EBV infected NPC mir-bart5 Cell survival

19 19 EBV+ EBV- Chemicaltriggered apoptosis apoptosis apoptosis TUNEL assay Apoptotic molecular marker PARP PARP be processed during apoptosis be degraded during apoptosis termini labeling

20 EBV-encoded mir-bart5 represses to protect the host from death 20 EBV- (BART5-) EBV+ (BART5+) C666-1 is EBV+ cell line Anti-BART5 EBV+ etoposide PARP PARP si EBV- EBV+ PARP PARP endogenous mir-bart

21 SUMMARY 21 p53 mir-bart5 Luciferase gene Cell death cell death rate Full length 3 UTR EBV+ EBV- Chemicaltriggered apoptosis High Low EBV-encoded mir-bart5 protects host from apoptosis by repressing

22 22 Thanks for your attention and professor Chang s advise

23 23 Conclusion and significance mir-bart5 mir-bart5

24 Small RNA regulators: RNA 24

25 Small RNA regulators: RNP 25

26 sirisc 26

27 Limitation of sirisc an example of TAR in HIV O-methyl oligonucleotide clamps

28 mirisc 28

29 29 Overview: biosynthesis and function of mirna

30 30 Possible inhibition model of mirna Initiation complex mirna may inhibit initiation, elongation, or induce mrna degradation inhibition complex

31 Molecular pathway of apoptosis 31

32 32 p53 network Cellular mirna Bcl-2 family Contains BH3 domain to neutralize BCL2 guard

33 Death and survival balance 33 Apoptotic protein v.s. survival proteins

34 Displacing Bak or Bax from its guards induces apoptosis 34

35 BH3 (Bcl-2 homology 3) domain 35 BH3 only v.s. BID BID is a pro-apoptotic Bcl-2 protein containing only the BH3 domain. In response to apoptotic signaling, BID interacts with another Bcl-2 family protein, Bax (Bcl-2-associated X protein), leading to the insertion of Bax into organelle membranes, primarily the outer mitochondrial membrane. Bax is believed to interact with, and induce the opening of the mitochondrial voltage-dependent anion channel, VDAC. Alternatively, growing evidence suggest that activated Bax and/or Bak (Bcl-2 homologous antagonist killer) form an oligomeric pore, MAC (the mitochondrial-induced apoptosis channel) in the outer membrane. This results in the release of cytochrome c and other proapoptotic factors from the mitochondria, often referred to as mitochondrial outer membrane permeabilization, leading to activation of caspases. This defines BID as a direct activator of Bax, a role common to some of the pro-apoptotic Bcl-2 proteins containing only the BH3 domain.

36 Potential targets of mir-bart5 predicted by miranda and RNAhybrid 36

37 Expression of mir-bart5 in EBV-infected tissues and cells NPC xenografts 37 Immortalized NP cell lines Primary NPC cell lines NPC xenografts Primary NPC cell lines EBV-infected Burkitt s lymphoma cell lines Primary NPC cell lines

38 Comparison of 3 UTR activity in HK1 and HK/EBV cells 38

39 Influence of anti-mir-bart5 on 3 UTR activity in C666-1 cells 39

40 40 Inhibition of mir-bart5 results in apoptosis

41 mir-bart5 and its target in different species 41

42 The clinical sampling 42

43 Down-regulate in mrna level 43 HEK293 Plasmid co-transfection

44 Etoposide and si activity test 44

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