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Mammalian RNAi pathways
Michael T. McManus
MIT Center for Cancer Research
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
RNAi: a tool for inhibiting gene expression in vivo
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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
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
How does RNAi work?
RNAi works postranscriptionally……..
in key two steps!
step one:
processing the dsRNA into 21-23 nt fragments
QuickT ime ™an d a
GIF deco mpre ssor
ar e need ed to see this pictur e.
34
27
21
20
16
short-interfering RNA
Tuschl, 2001
Dicer contains two RNAse III domains
long dsRNA
siRNAs
siRNAs have a defined structure
19 nt duplex
2 nt 3’ overhangs
step two:
the antisense strand of the siRNA guides cleavage
Tuschl, 2002
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
Model for RNAi
siRNA
Mammals exhibit potent responses to dsRNA
P P
dsRNA
PKR
interferon
production
P
eiF2a
apoptosis
Blockage of protein synthesis
cell
death
smaller RNAs can escape the PKR pathway
recall that siRNAs are
intermediate effectors
In the RNAi pathway
siRNAs are not recognized
by the PKR!
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?
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
develop an assay quantitative on the single-cell level
CD8
T-cell
CD4
flow
cytometry
detector
fluorescent antibodies detect
expression on the single cell level
transfection of plasmids and siRNAs
McManus, 2002
can any region of the mRNA be targeted with siRNAs?
CD8 mRNA
5’ UTR
CD8 ORF
3’ UTR
CD8 siRNAs
+
CD4 mRNA
5’ UTR
CD4 ORF
CD4 siRNAs
3’ UTR
+
McManus, 2002
Influenza mRNA target-sites
5’
3’
PB2
PB1
PA
NP
M
NS
UTR
Coding sequence
siRNA
No inhibition
Partial inhibition
Strong inhibition
Ge, 2003
how long does the RNAi response last?
% cells silencing CD8
12 0.00
10 0.00
80 .00
60 .00
40 .00
20 .00
0.00
0
10
10 00
10 0000
cell mass
McManus, 2002
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
establishing long-term RNAi
Let the cell make the siRNA for you!
CD8 hairpin RNAs
McManus, 2002
hairpin siRNAs
McManus, 2002
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
lentiviral construct for siRNAs
Rubinson et al Nature
Genetics, 2003
Lentiviral CD8 knockdown
Rubinson et al Nature
Genetics, 2003
stable 14-fold CD8 knockdown by lentivirus siRNAs
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
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