L’ARN interférence

Chantal VauryGReDUMR/CNRS6247, Clermont Université, INSERM U931Clermont-Ferrand

Introduction générale

Le principe de conservation

Il existe chez tous les êtres vivants des mécanismes communs telsque la production d’énergie, la réplication de l’ADN ou letransport des molécules.

Il est par conséquent possible d’étudier ces processus en utilisantdes organismes modèles faciles à manipuler.

Ces processus étant conservés entres les espèces, les résultatsobtenus avec le modèle seront transposables à d’autres êtresvivants tels que l’homme.

Concernant les eucaryotes, il existe des modèles phares tels quela levure, le nématode, la drosophile et la souris.

Le principe de conservation

Historique de

l’ARN interférence

Le nématode: Caenorabditis elegans

Antisense RNA

“Surprisingly, injection of sense ZC22 RNA also induced par-1 phenotypes. It is not clear what accounts for this effect… The basis for the sense effect is under investigation and will not be discussed further…”

Molecule Injected % embryonic lethality

ZC22 (par-1)antisense 52

ZC22 (par-1)sense 54

H20 0

“antisense” experiment Cell 81, 1-20 1995

RNA polymerases, although highly specific, produce some random or

ectopic transcripts

Nature 391, 806 1998

Molecules Injected % twitching

unc-22 “pure” antisense 0

unc-22 “pure” sense 0

unc-22 sense + antisense 100

Bacteria expressing

dsRNA GFP-expressing worms….

….eating regular bacteria

….eating bacteria

expressing GFP dsRNA

RNAi by ingesting dsRNA! - 1998Timmons and Fire - Nature 295, 854

Screen for RNAi-deficient or rde mutantsTabara et al., 1999 Cell 99, 123

Craig Mello

rde-1 is evolutionarily conserved!Tabara et al., 1999 Cell 99, 123

Attempts to deepen the purple hue of petunias by

genetic modification produced unexpected results.

Rather than heightening pigmentation, an inserted gene

switched colour production off, creating variegated

blooms (inset).

(Taken from Nature 404, 804 - 808)

Inactivation épigénétique post transcriptionnelle

PTGS and RNAi - a connection?Both are RNA-based gene silencing mechanisms

David Baulcombe

“Posttranscriptional gene silencing occurs in plants and fungi transformed with foreign or endogenous DNA and results in the reduced accumulation of RNA molecules with sequence similarity to the introduced nucleic acid.

Double-stranded RNA induces a similar effect in nematodes, insects, and protozoa.”

Hamilton and Baulcombe, 1999. Science 286, 952

Découverte de petits ARN complémentaires des cibles

Ces ARN sont appelés small interfering RNA ou siRNA

Small interfering RNA pathways

- si RNAs- mi RNAs- pi RNAs- endosi RNAs

Small interfering RNA pathway

RISC (RNA Induced Silencing Complex)Uses the siRNAs as guides to cleave target mRNAs

DICERCleaves the long dsRNA to make siRNAs

Le RISC

Molecular biology central dogma

DNA

RNA

Protein

Cellular function

Transcription

Translation

RNAi pathway

Des siRNAs aux miRNAs

Un monde de petits ARNs régulateurs

Cullen SR, 2005, Nature Genetics, 1163

miRNANaturallyoccuring small RNA

Molecular biology central dogma

DNA

RNA

Protein

Cellular function

Transcription

Translation

miRNAi pathway

microRNAs: “hetero-silencing”* Encoded in genome as hairpin precursors* Processed by DICER into 21-25nt RNAs* Targets loci distinct from the microRNA loci.* Degrade mRNAs with perfect match. * Block translation of mRNAs with imperfect match

siRNAs: “auto-silencing”* Agents for RNAi * 21-25nt RNAs processed by DICER (in some animals a different DICER gene than that used for microRNAs) from exo or endogenous

dsRNAs* Targets are RNAs from same or very similar locus * Degrade RNAs with perfect match

Des siRNAs et miRNAs

aux piRNAs ou rasiRNAs

Un monde de petits ARNs régulateurs

 Characteristics of Drosophila piRNAs

Drosophila piRNAs Map to Discrete Genomic Loci

The ping-pong model

Représentation schématique d’une région génomiquegénérant un cluster de piRNAs

Vagin et al., 2006, Science, 313, 320-324Carthew, 2006, Science, 313, 305-306.

Endo siRNA

Okamura and Lai, 2008NAT: Natural Antisens Transcripts

Exo-siRNA and Endo siRNA

Silencing transcriptionnel en lien avec le

silencing post-transcriptionnel

Immunité intrinsèque et « silencing »

Bloquer l’expression

Empêcher la transcription

TGS ou silencing transcriptionnel

Structure de la chromatine

Réduire la stabilité du messager

Bloquer sa traduction

PTGS ou silencing post-transcriptionnel

L’interférence ARN…

Chromatin packaging into chromosomes

Structure de la chromatineet silencing transcriptionnel

Le code histone

Ensemble de modifications chimiques de la chromatine à déchiffrer…

RNAi et silencing transcriptionnel sont parfois liés (S. pombe)

Chromatine inactivehétérochromatine

Chromatine activeÉléments répétés

RNAi

HP1/SWI6

HMT

HDAC

H3K9met

H3 H3

ADN méthyl-transférases

Méthylation de l’ADN

déacetylation

Chromatine

RITS

Volpe TA, et al., 2002, 297, 1833

Les séquences hétérochromatiques répriment leur propre expression.

Les séquences hétérochromatiques peuvent réprimer en trans l’expression d’une séquence qui leur est homologue.

Meth H3Lysine42

6

4

Meth H3Lysine 90.25

0.75

0.5

wt

Dcr

1-R

dp1-

Ago

1-

La voie d’ARN interférence guide le silencing de l’hétérochromatine

URA+

Tandemrepeats

Silencing en cis

La voie d’ARN interférence initie le silencing de l’hétérochromatine

RNAi et silencing transcriptionnel sont parfois liés

E

E

E

Propagation d’une structure chromatinienne compacte

Transitivité

Ou

Comment amplifier le signal?

Targeting GFP abolishes fluorescencebut also creates an unexpected, uncoordinated

phenotype.

Unc GFP

This occurs because of the production of double-stranded RNA and consequently small interfering RNAs

homologous to the endogenous UNC-22 gene.

Unc GFP

Production de trans-siRNAs

Systémie

ou comment distribuer le signal

Dispersion de l’ARNi à travers l’organisme

A silencing signal moves from

the veins into leaf tissue. Green = GFP fluorescence

Red = chlorophyll fluorescence

that is seen upon silencing of the GFP

transgene.

C. elegans engineered to express GFP in nuclei. Animals on the right have been treated

with a control dsRNA. Animals on the left have been exposed to GFP dsRNA.

Some neuronal nuclei remain fluorescent, correlating with low expression of a protein

required for systemic RNAi59.

Transitivité et systémie assurent l’efficacité du contrôle par la voie RNAi

RNAi cellular function

TC1

unc-22(-) unc-22(+)

transposition

In some rde mutants, transposition is increased

Transposable elements mobilisation is controlled by the RNAi pathway

Post-transcriptional inactivation in plantsallows résistance against viruses

In Arabidopsis thaliana mutants from the PTGS pathway

Are sensitive to viral infections

Viral siRNAs are found in resistant plants

Hamilton et Baulcombe, Science, 1999

RNAi pathway plays a role in development

MicroRNAs regulate brain morphogenesis in zebrafish.

Giraldez et al (2005), Science 308:833

MZ dicer: mutant for Dicer, necessary to mature miRNA.miR-430: miRNA which contributes to tissue-specific gene expression

- Fragile X Mental Retardation due to loss of FMRP- FMRP binds RNAs, regulates protein translation at synapse, and is thought to regulate the expression of specific genes during neural development - FMRP fly ortholog is subunit of RISC complex

MicroRNAs likely play role in some neurological diseases

Fragile X

Tourette syndrome

- Slit and Trk-like 1 (SLITRK1) : candidate gene- Frameshift mutation in the binding site for microRNA hsa-miR-189 - Abelson et al., Science. 2005, 310, 317-20.

Using RNAi

in the Prevention of Disease

Polyglutamine

-Amyloide

Toxic fold

Many neurodegenerative diseases are caused by toxic property of mutant proteins

Applying RNA interference to neurological diseases

A simple view of poly-Q diseaseHuntington disease

Normal CAG repeat

Normal Q repeat

Normal protein folding

Normal biological activity

Expanded CAG repeat

Expanded Q repeat

Protein misfolding

Toxic property

Xia H. et al., Nature Biotech., 2006

RNAi successfully acts against

preformed polyglutamine aggregates

Targeting viruses

VIRUS : HIV, Polyovirus, HPV, RSV, Hepatitis C

Traitement actuels : - drogues anti-virales mais variants résistants, toxicité- siRNAs contre ARN viral mais problème de variants résistants.

Solutions possibles: - Cibler les cofacteurs cellulaires nécessaires au virus- Présence systémique de longue durée et en quantité suffisante: transduction de cellules souches hématopoietiques

Viruses can counteract the cellular defense : Viral silencing suppressors

Suppresseurs de RNAi chez les plantes :Qu F, Morris TJ: FEBS Lett 2005, 579: 5958-5964

- P1/HC-Pro codés par les potyvirus: Affecterait l’assemblage

ou le ciblage du complexe RISC

- Suppresseur 2b du virus mosaic du concombre: Bloque le

mécanisme qui empêche l’invasion virale en bloquant la

systémie.

- p19 des tombus virus: s’associe aux siRNAs.

Suppresseurs de RNAi chez les animaux:Li HW, Ding SW. FEBS Lett 2005, 579: 5965-5973.

- B2 codé par le Flock house virus (FHV):supprime le silencing

dépendant de la voie Ago2

- Tat du HIV: s’associe à dsRNAs

- Tas du Primate Foamy Virus (PFV)

Libraries of siRNA's have been generated to silence a vast number of genes

in a high-throughput manner.

Such applications should become more common in the future,

particularly in a target-discovery context of drug-design, for example.

A Resource for Large-scale RNA-interference-based Screens