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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rote

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

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