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.
Mammalian RNAi pathways
Michael T. McManusMIT 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 andthen introduced into cells
• any mRNA with high sequence homology to the dsRNA may be silenced
nucleus
dsRNA
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
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!
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
Dicer contains two RNAse III domains
siRNAs
long dsRNA
siRNAs have a defined structure
19 nt duplex
2 nt 3’ overhangs
Tuschl, 2002
the antisense strand of the siRNA guides cleavagestep two:
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
siRNA
Model for RNAi
Mammals exhibit potent responses to dsRNA
PKR
PP
P
eiF2
dsRNA
Blockage of protein synthesis
interferonproduction
cell deathapoptosis
smaller RNAs can escape the PKR pathway
siRNAs are not recognized by the PKR!
recall that siRNAs are intermediate effectorsIn the RNAi pathway
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?
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
T-cellCD4
CD8flow
cytometrydetector
fluorescent antibodies detect expression on the single cell level
develop an assay quantitative on the single-cell level
transfection of plasmids and siRNAs
McManus, 2002
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
PB2
PB1
PA
NP
M
NS
UTR
5’ 3’
Coding sequence
No inhibition
Partial inhibition
Strong inhibition
siRNA
Influenza mRNA target-sites
Ge, 2003
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
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
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:
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
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
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