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

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 dsRNA nucleus •any mRNA with high sequence homology to the dsRNA may be silenced

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. 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 practical aspects of RNAi

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

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

7. Dicer contains two RNAse III domains long dsRNA siRNAs

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

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

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. Model for RNAi siRNA

12. P P interferon production PKR eiF2a P apoptosis Blockage of protein synthesis Mammals exhibit potent responses to dsRNA dsRNA cell death

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

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

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

17. transfection of plasmids and siRNAs McManus, 2002

18. CD8 mRNA 5’ UTR CD8 ORF 3’ UTR CD8 siRNAs CD4 mRNA 5’ UTR CD4 ORF 3’ UTR CD4 siRNAs can any region of the mRNA be targeted with siRNAs? + + McManus, 2002

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

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

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

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: Brummelkamp et al: Science Yu et al: PNAS Miyagishi et al: Nature Biotech Sui et al: PNAS Sook Lee et al: Nature Biotech Zeng et al: Mol Cell Paddison et al: Genes Dev Paul et al: Nature Biotech McManus et al:RNA

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