DOI: 10.1126/science.309.5740.1518a , 1518a (2005); 309Science

Poster: RNA Silencing

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RNAse IIIdsRNA-specific nucleases

ArgonauteCore proteinSome with catalytic activity

Neurospora crassaTetrahymenaVertebrateMouse

Fruit flyDrosophila melanogaster

NematodeCaenorhabditis elegans

Fission yeastSchizosaccharomyces pombe

PlantPetunia, Tobacco, Arabidopsis thaliana

HelicasesDestabilize dsRNAs

RdRpRNA-dependent RNA polymerases

dsRNA binding proteinsRNAse III associated

Others

Ago1RDE-1 (RNAi)

ALG-1 and ALG-2 (miRNA)

PPW-2 (transposon silencing)

PPW-1 (germline RNAi)

Ago1Ago2 (endonuclease)

Ago3Ago4

DCR-1 (RNAi, miRNA)Drosha (miRNA)

DRH-1/DRH-2 (RNAi)

SMG-2 (RNAi)

MUT-14 (transposon silencing,

germline RNAi)

RRF-1 (somatic RNAi)

QDE-2 (Quelling)

Aubergine (Stellate silencing,

heterochromatin silencing)

piwi (heterochromatin, cosuppression)

Ago1 (miRNA)

Ago2 (siRNA)

EGO-1 (germline RNAi)

RRF-3 (RNAi silencer)

RDE-4 (RNAi)

R2D2 (siRNA)

Pasha (pre-miRNA)

Loquacious (miRNA)

ERi-1 (RNAi)

RDE-3 (RNAi)

SID-1 (RNA transporter, RNAi)

MUT-7 (cosuppression, germline RNAi)

MUT-8 (transposon silencing, RNAi)

VIG-1 (miRNA)

TSN-1 (miRNA)

QDE-1 (Quelling)

QDE-3 (Quelling)

AGO1 (miRNA, PTGS*)

SGS2/SDE1/RDR6 (virus, PTGS*)

SDE3 (VIGS, PTGS*) Hrr1

Spindle-E (Stellate silencing,

heterochromatin)

Armitage (RISC formation)

Dcr1

Rdp1

Cid12

Dicer (RNAi and miRNA,

essential)

Drosha (miRNA)

Tudor-SN (RNAi and

miRNA) VIG (RNAi and miRNA)

FXR (RNAi and miRNA)

Tudor-SN (RNAi and miRNA)

AGO4 (heterochromatin)

DCL1 (miRNA)

DCL2 (virus related)

DCL3 (heterochromatin)

Hyl1 (miRNA)

DRB4

RDR1 (virus PTGS*)

*PTGS, posttranscriptional gene silencing

RDR2 (chromatin silencing)

DGCR8TRBP

Dicer-2 (RNAi)

Dicer-1 (miRNA)

Drosha (miRNA)

Gemin3 (miRNP)

(miRNA)

HEN1 (miRNA, RNA methylase)

Twi1

SGS3 (PTGS*)

SDE4/PoI IV subunits (RNAi) Exportin-5 (miRNA

transporter)

Gemin4 (miRNA)

Dicer (DCL1)

HASTY (miRNA transporter)

Pdd1

Twi1

Transcription of the genome

dsRNA

Exportation of scnRNAs into old macronucleus

Degradation ofscnRNAs with homology

Migration of remaining scnRNAs intonew macronucleus

Micronucleus

scnRNAs

Twi1

Twi1 Twi1

MeMe

Histone H3–lysine 9 methylation

The ciliated protozoan Tetrahymena

contains two nuclei. During somatic

growth, the germline micronucleus is

transcriptionally silent. About 15% of

the micronuclear genome consists of

germline-specific sequences (yellow) that are

eliminated when the somatic macronucleus is

formed. All gene expression occurs in the macronucleus

from the macronuclear-destined sequences (blue).

To accomplish this elimination, Dcl1 cleaves dsRNA molecules

formed by the bidirectional transcription of the micronuclear

genome into small “scan” RNAs (scnRNAs). The scnRNAs form

complexes with the Argonaute protein Twi1 and are transferred

into the old macronucleus, where scnRNAs with homology to

the macronuclear genome are degraded.

The remaining complexes, derived from micronuclear-specific

sequences, are exported to the new macronucleus where they target

methylation to their homologous sequences, which, in turn, recruits

Pdd1. The germline-specific sequences are then removed from the

macronucleus, leaving only macronuclear-specific sequences (blue).

Dicer

Pdd1

Ago1

Chp1

Tas3

Tas3

In plants, TGS often involves

DNA methylation. siRNAs are

implicated in the de novo

methylation of gene

sequences—including

non-CG methylation and CNG

methylation by different

complexes.

In fruit flies, RNAi-associated

genes such as piwi and

aubergine (two Argonaute

proteins) and spindle-E (an

RNA helicase) are involved in

TGS. Mutation in these genes

results in a loss of hetero-

chromatin. Piwi also has a

central role in cosuppression

in flies.

In fission yeast, the RNA-induced

transcriptional silencing (RITS) complex

contains centromere-specific siRNAs, an

Argonaute protein (Ago1), a chromodo-

main protein (Chp1), and Tas3.

RITS associates with chromatin

through siRNA-nascent transcript

base pairing, as well as binding to

H3–lysine 9 methylated nucleosomes. Clr4

RITScomplex

Once bound to chromatin, RITS

recruits another protein complex

that contains RdRP and two other

RNA-modifying enzymes (Cid12,

Hrr1). Together these proteins

initiate and maintain the silent

heterochromatin packaging on

centromeric regions of the

chromosomes.

Chp1

Tas3Me

Me

Chp1

Hrr1Cid12

RdRp

ViraldsRNA

Viral RNA

ViralsiRNA

Potyvirus

Ago

Ago

Blocks RISC complex formation

Blocks RISCactivityHc-Pro

Plant and likely animal cells direct siRNAs

against invading viruses to prevent or slow viral

replication. In the ongoing arms race between

host and pathogen, many plant and some

animal viruses in turn have evolved proteins,

suppressors of gene silencing, which interfere

with different steps of the RNAi machinery.

Ago

Hc-Pro, the RNAi

suppressor of the

Potyvirus family, which

infects plants, blocks

RISC activity rather than

affecting RISC formation.

The best-characterized silencing suppressor is

the p19 protein (above) of the Tombusvirus

family, which infects plants. Homodimerized

p19 sequesters siRNA, preventing the

formation of active RISC and consequent

silencing.

Tombus-virus

p19

p19

12

Dicer

MicroRNAs are encoded in the genome and produced by the maturation of

a hairpin-shaped RNA transcript. These RNAs are key regulators of many

biological processes such as development (see example in Zebrafish at

top right), cell proliferation, apoptosis, morphogenesis, oncogenesis (see

example in mouse at bottom right), and hematopoiesis. In animals, they

generally work by blocking protein synthesis or destabilizing mRNA. In

contrast, miRNAs from plants and some from animals predominantly

induce mRNA degradation and are important for plant siRNA formation.

They also act as defense mechanisms by targeting RNA from viruses,

leading to silencing of viral protein expression. MicroRNAs produced

by viruses can also affect expression of host genes.

Ventral view of mir-140 expression in Zebrafish embryos.

Overexpression of mir-17-19b clusters accelerates lymphomas formation in mice.

10

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Bacteriaexpressing

dsRNA

F1generation

F2generation

Virus

ViraldsRNA

HostsiRNAs

SDE3

SID-1

C. elegans can take up dsRNAs by ingestion of

bacteria expressing dsRNA molecules (which

requires a cellular factor called SID-1); by

being soaked in a solution containing dsRNA;

or by being injected with dsRNA into the body

cavity. Gene silencing spreads to most cells of

the animal, with the exception of neurons.

Moreover, RNA silencing can be passed on for

several generations. The physiological role of

spreading in C. elegans is unknown.

RNA silencing in plants

serves as an antiviral

mechanism. Viral infection

produces double-stranded

RNA (dsRNA) intermediates,

which are processed by the

host RNAi machinery into

small interfering RNAs

(siRNAs). Spreading of the

silencing signal throughout

the plant ensures that, when

the plant is later exposed to

the same virus, it will be

resistant to further infection.

In plants there are two types

of spreading: short-range

transmission of siRNAs from

cell to cell (involving SDE3)

and long-range transmission

of an unknown substance

through phloem.

TriggerSu(Ste) on Y chromosome

Tandem repeats

TargetStellate on X chromosome

Tandem repeats

dsRNA

rasiRNAs25–27 nt

Aubergine?

TGS?

mRNAcleavage

?

RISCcomplex

In the testis of male fruit flies, overexpression of the repetitive Stellate genes on the X chromosome is prevented by the

Suppressor of Stellate [Su(Ste)] locus on the Y chromosome. Overlapping sense and antisense RNAs are generated from

the Su(Ste) locus, which form dsRNAs. Dicer processes these dsRNAs into short repeat-associated siRNAs (rasiRNAs), with

the possible assistance of two RNA helicases Spindle-E and Armitage, and the dsRNA binding protein Loquacious. The

rasiRNAs are probably incorporated into a RISC-like complex with Aubergine, an Argonaute homolog, which silences the

Stellate genes.

AAAAA

A

Dicer

RNAi Suppressors VIRUSES STRIKE BACKSome viruses have evolved endogenous proteins that interfere with the gene silencing machinery, as a countermeasure to attenuate host antiviral defenses.

MicroRNAs MICROCONTROLLERS OF MULTIPLE PATHWAYSThese noncoding genes, found in nearly every eukaryotic organism, are often highly conserved through evolution and are involved in regulation of a diverse range of biological pathways.

DNA Elimination RNA-DIRECTED GENOME SHREDDINGIn this unusual process that may function as a germline defense mechanism,small “scan” RNAs generated by an RNAi-like process direct the elimination of DNA segments.

Stellate Silencing RNAi DOIN’ IT NATURALLYSilencing of the repetitive Stellate genes in Drosophila melanogaster, which isnecessary for male fertility, was the first example of endogenous dsRNA-mediated gene repression.

Small RNAs from both inside and outside the cell are processed by theRNA interference (RNAi) machinery to inhibit genes and proteins by cleavingmessenger RNAs, blocking protein synthesis, or inhibiting transcription.

CleavageDicer cleaves long

dsRNA into 21 - to 27- nucleotideintervals processively.

RISC loading complexOne strand of the siRNA duplex is loaded

into the RISC complex. The less complementary, and thus less stable, 5’ end will unwind more

easily. That strand will be incorporated into RISC. The other strand is eliminated.

RISC complexThe RNA-induced silencing complex (RISC) is the central element of all RNA

silencing pathways. It contains at least one Argonaute protein and a small noncoding RNA. This complex carries out one of three silencing operations, as dictated by

its specific RNA: mRNA cleavage, protein synthesis block, or transcriptional gene silencing (TGS).

siRNA fully complementary

to mRNAsiRNA partially

mismatched with mRNA

TGS

Protein synthesis block

miRNAs cooperatively bind to 3’ untranslated region (3’UTR) elements with imperfect complementarity and

prevent translation or destabilize the mRNAs.

RISC-induced sequence-specific cleavage of the target mRNA has been

found in plants, Drosophila, and mammals. Requirements are perfect complementarity and

catalytically active Argonaute (Slicer).

Messenger RNA cleavage

Crystal structure of the Argonaute protein with siRNA (red) and mRNA (green) inserted by model building. RNA-binding PAZ domain, blue; nuclease PIWI domain, purple.

RNASILENCING

Tetrahymena

PlantFruit flyFission yeast

Transcriptional Gene Silencing (TGS)CONTROL OF THE CHROMATIN STATESmall noncoding RNAs silence expression of genes with homologous sequences by preventing transcriptionat the DNA. TGS inhibits gene transcription by forming heterochromatin or promoting methylation.

Fruit fly

Spreading A PRIMITIVE “IMMUNE SYSTEM”RNA silencing can spread from cell to cell in plants and confer a sort of plant-wide immunity to viruses. In the nematode C. elegans, silencing can be transmitted from generation to generation.

Plant Nematode

Plant

Plant Nematode Mouse Fruit fly

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Viral RNAViruses can produce RNAsto hijack the RNA silencing pathways in the infected cell, furthering the viruses’ survival.

Scientist-supplied RNAExogenous RNA can be taken up intocells where it interferes with geneexpression through the endogenoussilencing pathways. This technique hasproven to be a powerful method ofinhibiting and testing gene function inmany organisms.

MicroRNAs (miRNAs)After transcription by RNA poly-merase, the folded pri-miRNA isprocessed at the 5’ end by Drosha toproduce pre-miRNA. The pre-miRNA istranslocated into the cytoplasm byexportin-5, where Dicer will completeits maturation into miRNA.

RNA from repetitive DNAor aberrant RNA

In plants and nematodes, transposons and transgenes, which contain repetitive DNA, encode double-stranded RNA through bi-directional transcription or RdRp activity. Theresulting siRNAs are used in TGS to inhibit geneexpression from the original DNA. Centromericheterochromatin and the mating type locus in fission yeast are also silenced in this way.Aberrant RNA is similarly processed and eliminated.

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