RNAi Mechanism. The Central Dogma DNA (double-stranded) RNA (single-stranded) Protein.

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RNAi Mechanism

Transcript of RNAi Mechanism. The Central Dogma DNA (double-stranded) RNA (single-stranded) Protein.

Page 1: RNAi Mechanism. The Central Dogma DNA (double-stranded) RNA (single-stranded) Protein.

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

r p

rote

inle

vel

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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