1. Mammalian RNAi pathways Michael T. McManus MIT Center for Cancer Research

2. what is RNA interference? RNAi is a way to silence gene expression to perform RNAi, dsRNA homologous to the targeted gene is made and then introduced into cells any mRNA with high sequence homology to the dsRNA may be silenced nucleus dsRNA

3. RNAi: a tool for inhibiting gene expression in vivo C. elegans (Fire et al., 1998) Drosophila (Carthew et al., 1998) Planaria (Newmark et al., 1998) Trypanosomes (Ullu et al., 1998) Hydra (Lohmann et al., 1999) Zebrafish (Wargelius et al., 1999) Mice (Wianny & Zernicka-Goetz, 2000) “cosuppression” in plants “quelling” in Neurospora

4. practical aspects of RNAi biological research –defining gene function (gene knockout) C. elegans genome RNAi projects –defining biochemical pathways microarray screening of RNAi knockouts therapeutic treatment –cancer –viral infection –parasitic infection

5. How does RNAi work? RNAi works postranscriptionally…….. in key two steps!

6. processing the dsRNA into 21-23 nt fragments 34 27 21 20 16 short-interfering RNA step one: Tuschl, 2001

7. Dicer contains two RNAse III domains siRNAs long dsRNA

8. siRNAs have a defined structure 19 nt duplex 2 nt 3 ’ overhangs

9. Tuschl, 2002 the antisense strand of the siRNA guides cleavage step two:

10. RNAi silencing complex may be associated with translating ribosomes active RNAse enzyme not yet identified may participate in endogenous pathways that silence genes via translational repression

11. siRNA Model for RNAi

12. Mammals exhibit potent responses to dsRNA PKR P P P eiF2  dsRNA Blockage of protein synthesis interferon production cell death apoptosis

13. smaller RNAs can escape the PKR pathway siRNAs are not recognized by the PKR! recall that siRNAs are intermediate effectors In the RNAi pathway

14. need to further characterize mammalian RNAi how long does it last? how much dsRNA is required? can any region of a gene be effectively targeted?

15. T-cell how to get siRNAs into the T-cells electroporation receptor-dependent transport, endocytosis, etc. cationic lipids, calcium phosphate, etc. dead cells no silencing

16. T-cell CD4 CD8 flow cytometry detector fluorescent antibodies detect expression on the single cell level develop an assay quantitative on the single-cell level

17. transfection of plasmids and siRNAs McManus, 2002

18. 5’ UTR CD8 ORF3’ UTR CD8 siRNAs CD8 mRNA 5’ UTR CD4 ORF3’ UTR CD4 mRNA CD4 siRNAs can any region of the mRNA be targeted with siRNAs? + + McManus, 2002

19. PB2 PB1 PA NP M NS UTR 5’3’ Coding sequence No inhibition Partial inhibition Strong inhibition siRNA Influenza mRNA target-sites Ge, 2003

20. how long does the RNAi response last? cell mass % cells silencing CD8 McManus, 2002

21. miniconclusion RNAi via siRNAs is transient, lasting ~3-6 cell doublings RNAi creates knock-downs, not knockouts! not every siRNA works RNAi works by target degradation of the mRNA

22. establishing long-term RNAi Let the cell make the siRNA for you!

23. CD8 hairpin RNAs McManus, 2002

24. hairpin siRNAs McManus, 2002

25. stable mammalian RNAi Within a three month window: McManus et al:RNA Brummelkamp et al: Science Paul et al: Nature Biotech Sui et al: PNAS Yu et al: PNAS Sook Lee et al: Nature Biotech Miyagishi et al: Nature Biotech Paddison et al: Genes Dev Zeng et al: Mol Cell

27. lentiviral construct for siRNAs Rubinson et al Nature Genetics, 2003

28. Lentiviral CD8 knockdown Rubinson et al Nature Genetics, 2003

29. stable 14-fold CD8 knockdown by lentivirus siRNAs Rubinson et al Nature Genetics, 2003

30. functional silencing of genes in ES cell-derived mice by lentivirus-induced RNAi Rubinson et al Nature Genetics, 2003

31. mini-conclusion RNAi knock-down mice can be generated in <30 days RNAi silencing can be transmitted through the germline Although silencing by siRNAs is transient, vectors can be made to express siRNAs in cells