Mammalian RNAi pathways Michael T. McManus MIT Center for Cancer Research.
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Transcript of Mammalian RNAi pathways Michael T. McManus MIT Center for Cancer Research.
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Mammalian RNAi pathways
Michael T. McManusMIT Center for Cancer Research
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what is RNA interference?
• RNAi is a way to silence gene expression
• to perform RNAi, dsRNA homologous to the targeted gene is made andthen introduced into cells
• any mRNA with high sequence homology to the dsRNA may be silenced
nucleus
dsRNA
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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
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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
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How does RNAi work?
RNAi works postranscriptionally……..
in key two steps!
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processing the dsRNA into 21-23 nt fragments
3427212016
short-interfering RNA
QuickTime™ and aGIF decompressor
are needed to see this p icture.
step one:
Tuschl, 2001
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Dicer contains two RNAse III domains
siRNAs
long dsRNA
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siRNAs have a defined structure
19 nt duplex
2 nt 3’ overhangs
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Tuschl, 2002
the antisense strand of the siRNA guides cleavagestep two:
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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
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siRNA
Model for RNAi
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Mammals exhibit potent responses to dsRNA
PKR
PP
P
eiF2
dsRNA
Blockage of protein synthesis
interferonproduction
cell deathapoptosis
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smaller RNAs can escape the PKR pathway
siRNAs are not recognized by the PKR!
recall that siRNAs are intermediate effectorsIn the RNAi pathway
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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?
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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
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T-cellCD4
CD8flow
cytometrydetector
fluorescent antibodies detect expression on the single cell level
develop an assay quantitative on the single-cell level
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transfection of plasmids and siRNAs
McManus, 2002
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5’ UTR CD8 ORF 3’ UTR
CD8 siRNAs
CD8 mRNA
5’ UTR CD4 ORF 3’ UTR
CD4 mRNA
CD4 siRNAs
can any region of the mRNA be targeted with siRNAs?
+
+
McManus, 2002
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PB2
PB1
PA
NP
M
NS
UTR
5’ 3’
Coding sequence
No inhibition
Partial inhibition
Strong inhibition
siRNA
Influenza mRNA target-sites
Ge, 2003
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how long does the RNAi response last?
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 10 1000 100000
cell mass
% c
ells
sile
nci
ng
CD
8
McManus, 2002
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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
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establishing long-term RNAi
Let the cell make the siRNA for you!
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CD8 hairpin RNAs
McManus, 2002
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hairpin siRNAs
McManus, 2002
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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
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lentiviral construct for siRNAs
Rubinson et al Nature Genetics,
2003
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Lentiviral CD8 knockdown
Rubinson et al Nature Genetics,
2003
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stable 14-fold CD8 knockdown by lentivirus siRNAs
Rubinson et al Nature Genetics,
2003
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functional silencing of genes in ES cell-derived mice by lentivirus-induced RNAi
Rubinson et al Nature Genetics,
2003
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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