Transcript Document

RNAi Mechanism
The Central Dogma
DNA (double-stranded)
RNA (single-stranded)
Protein
When good RNA goes bad....
Viruses can
make doublestranded RNA
When good RNA goes bad....
Cells sense doublestranded RNA and
activate a response
called RNAi
Understanding how
RNAi works is the
key to using it as a
genetic tool and for
therapy
What is the mechanism of interference?
RNAi in vitro...
Cell free extract
RNAi?
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
RNAi functions in many different organisms
dsRNA
Dicer is required for RNAi
wild-type
GFP
dsRNA
GFP – germline transgene
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 Song
Leemor Joshua-Tor
PIWI is an RNase H like domain
Argonaute-PIWI
RNase HI
RNase HII
Ji Joon Song
Leemor Joshua-Tor
A model for siRNA directed cleavage
microRNAs regulate gene
expression without cleavage
RNA or protein
level
small temporal RNAs (stRNAs)
LIN-14, LIN-28 proteins
LIN-41 protein
lin-4 RNA
let-7 RNA
L1 stage
L2 stage
L3 stage
LIN-29 protein
L4 stage
Adult stage
• lin-4 miRNA represses translation of lin-14 (L1 to L2 moult)
stRNA precursor
Dicer
stRNA
Translational
repression
Banerjee & Slack, 2002
Ambros lab, Rukvun lab
Mello lab, Zamore lab
RNAi acts to regulate gene expression by two
dicer-dependent mechanisms
miRNA
siRNA
Model for translational repression
M7GpppG
RISC recognizes a target
RISC
AAAAAA.........
Model for translational repression
M7GpppG
DCP GW
RISC
Other proteins are also recruited –
either along with RISC or later
other
AAAAAA.........
Model for translational repression
decapping
to P-bodies
DCP GW
RISC
M7GpppG
other
block translation?
(e.g. Filipowicz)
AAAAAA.........
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-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
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
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