Faghihi, MA et al . Nature Medicine 2008 Presenter: Chen-Chung Lin

1. Expression of a noncoding RNA is elevated in Alzheimer’sdisease and drives rapid feed-forward regulation ofb-secretase Faghihi, MA et al. Nature Medicine 2008 Presenter: Chen-Chung Lin

2. Main Claims • A BACE1-antisense transcript may contribute to Alzheimer‘s disease by regulating BACE-1 mRNA and protein expression. • BACE1-AS could be upregulated by Ab1-42 and stabilized the BACE1 mRNA, further express more BACE1 proteins and produce more Ab1-42. • Stabilization mechanism is by forming a RNA duplex, and protect RNA from degradation. • BACE1-AS contribute to AD formation.

3. Introduction • Alzheimer‘s disease (AD): • Neurodegenerative disease • Most common form of dementia • Characterized by the progressive formation in the brain of insoluble amyloid plaques and vascular deposits consisting of the 4-kD amyloid b-peptide (Ab) • amyloid b-peptide (Ab) • Cleavage of amyloid precursor protein (APP) by b-secretase-1 (BACE1)

4. Alzheimer‘s disease (AD) Ab synthesis pathway Rate limiting step amyloid precursor protein (Adapted from wikipedia, http://en.wikipedia.org/wiki/Amyloid_beta)

5. Dysregulation of BACE1 and Ab production in Alzheimer's disease Adapted from Peter St George-Hyslop & Christian Haass Nature Medicine 14, 711 - 712 (2008)

6. It contains transcript variants It is highly conserved between species. Genomic organization of BACE1 and BACE1-AS Chr.11 (104 nts complete match)

7. Expression analysis of BACE1 and BACE1-AS BACE1 mRNA expression is about 25-75% greater than BACE1-AS

8. BACE1-AS regulates BACE1 mRNA expression in vitro BACE1-AS siRNA knockdown also see BACE1 mRNA knockdown; overexpressed BACE1-AS could also see BACE1 mRNA increased.

9. BACE1-AS regulates BACE1 protein expression in vitro BACE1-AS siRNA knockdown could find that BACE1 protein knockdown; overexpressed BACE1-AS could also see BACE1 protein increased.

10. Knockdown of BACE1-AS reduces Ab 1–40 and Ab 1–42

11. Knockdown of BACE1-AS reduces BACE1 levels in vivo Cerebellum (No siRNA reached by perfusion) Cortex Striatum D. hippocampus V. hippocampus

12. Cell stressors increase BACE1-AS and BACE1 protein

13. BACE1-AS forms RNA duplex and increases stability of BACE1

14. BACE1-AS is elevated in subjects with Alzheimer’s disease In patients, BACE1-AS elevated.

15. APP transgenic mice have increased levels of Bace1-AS

16. Conclusion • BACE1-AS is a mature non coding RNA. • BACE1-AS could protect BACE1 from degradation by forming a dsRNA structure, and increase BACE1 RNA stability. • Ab could induce both BACE1 and BACE1-AS, then cause AD.

17. Questions • Would BACE1-AS transcript undergo NMD? • Would RNAse H degrade BACE1 and BACE1-AS dsRNA?

18. Complex I Binding by a VirallyEncoded RNA RegulatesMitochondria-Induced Cell Death Reeves, MB et al, Science 2007

19. Claims • HCMV noncoding RNA b2.7 would associated with mitochondrial complex I and then protect cell from apoptosis.

20. Introductions • Human cytomegalovirus (HCMV): • DNA virus • Betaherpesvirus (b-HSV) • Contains the largest DNA genome (240 kb) of human herpes virus. • RNA transcripts contains coding and noncoding RNAs. • Common cause of infants infection • Cytomegalic inclusion disease (Adapted from Brooks, GF et al. Medical Microbiology, p401-405 )

21. Shutting down the immune response by HCMV Block the innate immunity of infected cell. (MICB, NK cell activator) (Adapted from Cullen, BR Science317:329, 2007 )

22. Retenone Cell Death Mitochondria electron transport chain (Adapted from wikipedia, http://en.wikipedia.org/wiki/NADH_dehydrogenase)

23. HCMV protects cells from rotenone-induced apoptosis HMCV -Db2.7 Revertant HMCV -Db2.7 HMCV (Tx b2.7 plasmid) Retenone (mitochondria complex I inhibitor) b2.7 transcript would protect U373 cell from retenone induced apotosis.

24. What are the protection mechanisms of HCMV b2.7 RNA? • May be to mediate protection of cell from apoptotic pathways activated by metabolic stress of complex I • GRIM-19?

25. GRIM-19 (Genes associated with retinoid/interferon-induced mortality) • A subunit of Complex I • Essential for Complex I assembly • Essential for Complex I function • Report as a cell death-regulatory gene under retinoid / interferon treatment • RNA level remain steady after HCMV infection

26. HCMV prevents the rotenone-inducedrelocalization of GRIM-19 in virally infected cells RED: GRIM-19

27. HCMV interacts with GRIM-19 in virally infected cells IP: GRIM-19, Complex-1 or Complex-V RT-PCR HCMV Viral RNA IP on biotin-labeled antisense of b2.7 or IE72 Western blot for GRIM-19 Indicated that b2.7 interact with GRIM-19 Sliver stain of gels

28. b2.7 expressionmaintains ATP production in infected cells. 2 x b2.7 Long term Short term

29. Toledo, but not Db2.7Tol, grows normally in metabolically stressed cells. Deletion of b2.7 gene has no significant effects on viral growth in human fibroblasts (HF).

30. Toledo, but not Db2.7Tol, grows normally in metabolically stressed cells. Deletion of b2.7 gene has significant decrease effects on viral growth in U373 cells.  b2.7 transcripts support viral growth Deletion of b2.7 gene has significant decrease effects on viral growth in glucose-depleted U373and HF cells. b2.7 transcripts support viral growth under normal tissue condition (w/o too much glucose)

31. Conclusion • HCMV noncoing RNA b2.7 could binding to GRIM-19 and maintain the ATP production from mitochondrial electron transport chain; prevent HCMV infected cell from apoptosis.

32. Questions • Other targets for b2.7 RNA?