RNAi Mechanism
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Transcript of RNAi Mechanism
RNAi Mechanism
The Central Dogma
DNA (double-stranded)
RNA (single-stranded)
Protein
When good RNA goes bad....
Viruses canmake double-stranded RNA
When good RNA goes bad....
Cells sense double-stranded RNA andactivate a responsecalled RNAi
Understanding howRNAi works is the key to using it as a genetic tool and fortherapy
Cell free extract
RNAi?
What is the mechanism of interference?RNAi in vitro...
Hannon Lab, Zamore Lab, Tuschl Lab, Sharp Lab
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.
Dicer is an evolutionarily conserved nuclease
dsRNA
RNAi functions in many different organisms
GFP – germline transgene
Dicer is required for RNAi
GFPdsRNA
wild-type
dcr1-/-
How do small dsRNAs turn genes off?
Dicer-cut siRNA is used as a template to direct cleavage of complementary mRNA by RISC
RISC: RNA-inducing silencing complex
Argonaute proteins lie at the heart of RISC
Ji-Joon SongLeemor Joshua-Tor
Argonaute-PIWI RNase HI RNase HII
PIWI is an RNase H like domain
Ji Joon SongLeemor Joshua-Tor
A model for siRNA directed cleavage
microRNAs regulate gene expression without cleavage
small temporal RNAs (stRNAs)
stRNA precursor
stRNA
Translational repression
RN
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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
siRNA miRNA
RNAi acts to regulate gene expression by two dicer-dependent mechanisms
Model for translational repression
M7GpppG AAAAAA.........RISC
RISC recognizes a target
Model for translational repression
M7GpppG AAAAAA.........RISC
DCP GW
other
Other proteins are also recruited – either along with RISC or later
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
Least well understood - Arabidopsis as a model….
DNA is embedded in chromatin
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
Model for siRNA-dependent initiation of heterochromatic silencing by RITS
RITS: RNA-induced initiation of transcriptional gene silencing complex
dsRNA-mediated silencing in various organisms:Multiple mechanisms that are Dicer-dependant
Meister & Tuschl, 2004
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