RNAi Mechanism
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Transcript of 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 canmake double-stranded RNA
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When good RNA goes bad....
Cells sense double-stranded RNA andactivate a responsecalled RNAi
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Understanding howRNAi works is the key to using it as a genetic tool and fortherapy
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Cell free extract
RNAi?
What is the mechanism of interference?RNAi in vitro...
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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dsRNA
RNAi functions in many different organisms
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GFP – germline transgene
Dicer is required for RNAi
GFPdsRNA
wild-type
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 SongLeemor Joshua-Tor
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Argonaute-PIWI RNase HI RNase HII
PIWI is an RNase H like domain
Ji Joon SongLeemor 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)
stRNA precursor
stRNA
Translational repression
RN
A o
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inle
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L1 stage L2 stage L3 stage L4 stage Adult stage
LIN-14, LIN-28 proteins LIN-41 protein LIN-29 protein
let-7 RNAlin-4 RNA
Ambros lab, Rukvun lab
Dicer
Mello lab, Zamore lab
• lin-4 miRNA represses translation of lin-14 (L1 to L2 moult)
Banerjee & Slack, 2002
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siRNA miRNA
RNAi acts to regulate gene expression by two dicer-dependent mechanisms
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Model for translational repression
M7GpppG AAAAAA.........RISC
RISC recognizes a target
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Model for translational repression
M7GpppG AAAAAA.........RISC
DCP GW
other
Other proteins are also recruited – either along with RISC or later
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Model for translational repression
M7GpppG AAAAAA.........RISC
DCP GW
other
decapping to P-bodies
block translation?(e.g. Filipowicz)
de-adenylation?(e.g. Giraldez, Belasco, Rivas)
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-independentlinker 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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Levine and Davidson, PNAS, 2005
Ke, et al.Current Opinion in Chemical Biology 2003
dsRNA-mediated regulation may play a central rolein the regulation of Gene Networks
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