An Epstein-Barr virus-encoded microRNA targets PUMA to promote host - PowerPoint PPT Presentation

1 An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival The Journal of Experimental Medicine 205(11): 2551-2560, 2008. Elizabeth Yee-Wai Choy, Kam-Leung Siu, Kin-Hang Kok, Raymond Wai-Ming Lung, Chi Man Tsang, Ka-Fai To, 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) Belongs to dsDNA γ -herpesvirus Capsid diameter: approx. 100 nm Host: Epithelial cells Chemical inducers (TPA, Na butyrate), (TPA, Na butyrate), B lymphocytes B lymphocytes Stress Life cycle: Latent phase Latent/lytic 2 stage Latent protein, miRNA Pathogenicity: 90% people was infected Hodgkin’s disease, Burkitt’s lymphoma, and nasopharyngeal carcinoma (NPC) Tumor Lytic phase

3 microRNA (miRNA) miR-BART5 ˙Small regulatory RNAs ˙~22 nt in length ˙miRNA gene or intronic miRNA ˙Target 3’ UTR sequence ˙Target 3’ UTR sequence ˙Most target Imperfectly ˙Both cellular and viral miRNA are found miR-BART5 miRNA 5’ UTR Gene 3’ UTR PUMA mRNA

4 Now-known microRNA of EBV miR-BART5 Intronic segments Previous known miRNA Latent promoters Latent genes Exonic segments Newly identified miRNA Lytic promoters Lytic genes EBER: EBV-encoded small RNAs BART: Bam HI-A region rightward transcript

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

6 The story is… EBV PUMA miR-BART5 � immortalization � � �

7 Specific aim Normally… PUMA DNA p53 Cell death signal, PUMA mRNA Stress, … Cell death PUMA PUMA DNA p53 EBV infection Latency ? PUMA mRNA miRNA ? Cell death signal, Cell survival PUMA Stress, … miR-BART5

8 Framework In silica prediction, confirming EBV infected NPC PUMA miR-BART5 Cell survival

9 Luciferase reporter assay 3’ UTR Luciferase gene miR-BART5 GAPDH - Complementary sequence in PUMA 3’ UTR in PUMA 3’ UTR 4 binding sites 4 binding sites (binding site) Binding site mutant Perfectly match + with miR-BART5 PUMA 3’ UTR inhibition Luciferase gene

10 miR-BART5 binds 3’ UTR of PUMA inhib ibition - mutant + Luciferase gene

11 PUMA is cellular target of miR-BART5 plasmid inhibition Luciferase gene Full length PUMA 3’ UTR Full length PUMA 3’ UTR Full length PUMA 3’ UTR Chemical synthetic Luciferase gene miRNA precursor Inverted PUMA 3’ UTR Anti-sense of Intact PUMA 3’ UTR PUMA 3’ UTR

12 Framework In silica prediction, confirming EBV infected NPC PUMA miR-BART5 Cell survival

13 Assumption PUMA infec EBV- ection EBV+ PUMA

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

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

16 exogenous BART5 PUMA PUMA infec EBV- ction EBV+ Anti-BART5 PUMA PUMA

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

18 Framework In silica prediction, confirming EBV infected NPC PUMA miR-BART5 Cell survival

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

20 EBV-encoded miR-BART5 represses PUMA to protect the host from death C666-1 is EBV+ cell line EBV- (BART5-) EBV+ (BART5+) etoposide EBV+ Anti-BART5 PARP PARP PARP PARP EBV- siPUMA PARP PUMA PARP PUMA EBV+ endogenous + + + miR-BART5

21 SUMMARY p53 PUMA Cell death miR-BART5 Luciferase gene Full length PUMA 3’ UTR cell death rate PUMA High EBV- Chemical- triggered EBV+ apoptosis Low PUMA EBV-encoded miR-BART5 protects host from apoptosis by repressing PUMA

22 Thanks for your attention your attention and professor Chang’s advise

23 Conclusion and significance miR-BART5 miR-BART5

24 Small RNA regulators: RNA

25 Small RNA regulators: RNP

26 siRISC

27 Limitation of siRISC an example of TAR in HIV 2’-O-methyl oligonucleotide clamps

28 miRISC

29 Overview: biosynthesis and function of miRNA

30 Possible inhibition model of miRNA Initiation complex miRNA may inhibit initiation, elongation, or induce mRNA degradation inhibition complex

31 Molecular pathway of apoptosis PUMA

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

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

34 Displacing Bak or Bax from its guards induces apoptosis

35 BH3 (Bcl-2 homology 3) domain • “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 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 pro- apoptotic 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

Immortalized NP cell lines EBV-infected tissues and cells NPC xenografts Expression of miR-BART5 in Primary NPC cell lines NPC xenografts Primary NPC cell lines EBV-infected Burkitt’s lymphoma cell lines Primary NPC cell lines 37

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

39 Influence of anti-miR-BART5 on PUMA 3’ UTR activity in C666-1 cells

40 Inhibition of miR-BART5 results in results in apoptosis

41 miR-BART5 and its target in different species

42 The clinical sampling

43 Down-regulate PUMA in mRNA level HEK293 Plasmid co-transfection Plasmid co-transfection

44 Etoposide and siPUMA activity test