1. Mammalian RNAi pathways MIT Center for Cancer Research
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. 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. RNAi works postranscriptionally……..
How does RNAi work?
RNAi works postranscriptionally……..
in key two steps!

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

7. Dicer contains two RNAse III domains
long dsRNA
siRNAs

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

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

13. siRNAs are not recognized
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. flow cytometry detector
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. + + can any region of the mRNA be targeted with siRNAs? CD8 mRNA
5’ UTR
CD8 ORF
3’ UTR
CD8 siRNAs
CD8 mRNA +
5’ UTR
CD4 ORF
3’ UTR
CD4 mRNA
CD4 siRNAs +
McManus, 2002

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

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

21. RNAi works by target degradation of the mRNA
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. Within a three month window:
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. Rubinson et al Nature Genetics, 2003
lentiviral construct for siRNAs
Rubinson et al Nature Genetics, 2003

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

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

30. Rubinson et al Nature Genetics, 2003
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