RNAi Mechanism

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RNAi Mechanism
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The Central Dogma
DNA (double-stranded)
RNA (single-stranded)
Protein
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When good RNA goes bad....
Viruses can make double- stranded RNA
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When good RNA goes bad....
Cells sense double- stranded RNA and activate a
response called RNAi
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Understanding how RNAi works is the key to using
it as a genetic tool and for therapy
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What is the mechanism of interference? RNAi in
vitro...
Cell free extract
RNAi?
Hannon Lab, Zamore Lab, Tuschl Lab, Sharp Lab
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Dicer cuts dsRNA into short RNAs
Vanhecke, D. Janitz, M. Drug Discov. Today,
2005, 10, 205-225.
Role for a bidentate ribonuclease in the
initiation step of RNA interference Emily
Bernstein, Amy A. Caudy, Scott M. Hammond
Gregory J. Hannon
2001 Bernstein et al. Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains.
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Dicer is an evolutionarily conserved nuclease
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RNAi functions in many different organisms
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Dicer is required for RNAi
wild-type
GFP dsRNA
GFP germline transgene
dcr1-/-
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How do small dsRNAs turn genes off?
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Dicer-cut siRNA is used as a template to direct
cleavage of complementary mRNA by RISC
RISC RNA-inducing silencing complex
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Argonaute proteins lie at the heart of RISC
Ji-Joon Song Leemor Joshua-Tor
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PIWI is an RNase H like domain
Argonaute-PIWI
RNase HI
RNase HII
Ji Joon Song Leemor Joshua-Tor
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A model for siRNA directed cleavage
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microRNAs regulate gene expression without
cleavage
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small temporal RNAs (stRNAs)
lin-4 miRNA represses translation of lin-14
(L1 to L2 moult)
Ambros lab, Rukvun lab
Mello lab, Zamore lab
Banerjee Slack, 2002
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RNAi acts to regulate gene expression by two
dicer-dependent mechanisms
miRNA
siRNA
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Model for translational repression
RISC
AAAAAA.........
M7GpppG
RISC recognizes a target
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Model for translational repression
GW
DCP
RISC
AAAAAA.........
M7GpppG
other
Other proteins are also recruited either
along with RISC or later
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Model for translational repression
decapping
to P-bodies
GW
DCP
RISC
AAAAAA.........
M7GpppG
other
de-adenylation? (e.g. Giraldez, Belasco, Rivas)
block translation? (e.g. Filipowicz)
RISC may block through multiple mechanisms
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Least well understood - Arabidopsis as a model.
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DNA is embedded in chromatin
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Regulation of gene expression at the level of
chromatin
Sequence-independent linker histones control
DNA compaction and accessibility to trans-acting
factors post-translational modifications of
histone tails control compaction of DNA and
serve as docking sites for trans-acting
factors Range Can act at the level of a single
gene, often acts over groups of genes and over
larger domains (20-200kb), and can affect gene
expression over an entire chromosome
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Model for siRNA-dependent initiation of
heterochromatic silencing by RITS
RITS RNA-induced initiation of transcriptional
gene silencing complex
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dsRNA-mediated silencing in various
organisms Multiple mechanisms that are
Dicer-dependant
Meister Tuschl, 2004
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dsRNA-mediated regulation may play a central
role in the regulation of Gene Networks
Levine and Davidson, PNAS, 2005
Ke, et al. Current Opinion in Chemical Biology
2003
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