UMass Caenorhabditis elegans Bob Goldstei ! Sydney Brenner David - - PowerPoint PPT Presentation

RNAi and Development in C. elegans

Craig C. Mello Stockholm December 10, 2006

UMass

Caenorhabditis elegans

Bob Goldstei!

Dan Stinchcomb Victor Ambros Jim Priess Sydney Brenner David Hirsh Bob Horvitz John Sulston

Nematophagous fungi

Arthrobotrys dactyloides Photo by George Barron

Arthrobotrys dactyloides can catch C. elegans Mark Alkema

Nova Science Now

DNA

Return to the RNA world

small RNAs & development Evolution?

Weismann’s theory (ala Ernst Mayr/& post RNAi). (1)There is a special particle (biophore) for each trait. (2)These particles can grow and multiply independent of cell division. (3)Both nucleus and cytoplasm consist of these biophores. (4)A given biophore may be represented by many replicas in a single nucleus, including that of a germ cell. (5)During cell division the daughter cells may receive different kinds and numbers of biophores (unequal cell division). “As we now know, postulates (2) and (5) are wrong and are responsible for the fact that Weismann was not able to arrive at a correct theory of inheritance.” RNA-directed inheritance?

siRNAs (with siRNAs) some s.

contain siRNAs.

siRNA siRNAs NOT WRONG.

RNA may play a role in inheritance and evolution.

cbs news explains RNAi

Nova Science Now Robert Krulwich Eric Lander

dsRNA targetting essential maternal genes

uninjected

carrier

• ocytes

unaffected embryos carrier

• ocytes

F1 F1 dead eggs

X

F2 affected dead eggs

F3

F1 carriers

P0

F2

rde/mut(+) rde/mut (-) rde/mut (-)

Heritable Systemic Amplified! RNA injection Potent Sequence-specific Carriers

RNA-dependent RNA-degradation Accumulation Accumulation of

• f RNAi

RNAi active active molecules molecules in in the the target target tissue tissue pre-mRNA

Catalytic model of RNAi

Injected dsRNA Degradation Transport Transport into into the the cells cells Target tissue

Foreign dsRNA Recognition Transport Silencing Amplification and Inheritance

guided

RNAi

P0 F2

Hiroaki’s screen for RNAi deficient mutants (1998-1999) Hiroaki Tabara

wt RNAi induced dead eggs RNAi-deficient viable progeny Feed dsRNA (Lisa Timmons) Mutagenize

Analysis of Hiroaki’s RNAi Deficient Mutants # alleles Other Phenotypes rde-2 3 !sterile/him/mutator rde-3 3 sterile/him/mutator rde-4 2 none rde-5 1 sterile/him/mutator rde-6 2 sterile/him/mutator rde-1 6 none mut-7 1 sterile/him/mutator mutator mutator mutator mutator

RNAi Transposons What about RDE-1?

Hypothesis: Related silencing pathways with distinct triggering mechanisms

Transgenes

Hiroaki Tabara Argonaute protein

PAZ PIWI

1020aa 334 443 618 985

RDE-1 PAZ PIWI

1020aa 334 443 618 985

RDE-1 Neurospora QDE-2 Quelling (transgene silencing

Cogoni and Macino, 2000

• C. elegans

RDE-1 RNAi

(Tabara et al., 1999)

Drosophila Aubergine/Sting Development and Epigenetic silencing

(Schmidt et al., 1999 Wilson et al., 1996)

Arabidopsis AGO1 Development Gene silencing

(Fagard et al., 2000 Bohmert et al., 1998)

RDE-1 Cloning reveals links to development and gene silencing in insects, plants and fungi!

Argonaute protein

ceRDE-1 ceZK1248.7 ceF58G1.1 cePPW-2 ceR06C7.1 ceF55A12.1 ceY49F6A.1 ceR04A9.2 ceT22H9.3 ceC16C10.3 ceM03D4.6 ceK12B6.1 cePPW-1 ceF56A6.1 ceF20D12.1 ceC04F12.1 ceR09A1.1 ceD2030.6 ceTAG-292 hsHILI hsHIWI2 hsPIWIL3 hsHIWI dmAUB dmPIWI dmAGO2 spAGO1 atAGO5 atZLL/PNH atAGO1 atAGO6 atAGO4 atAaGO9 atAGO7 atAGO2 atAGO3 ceT22B3.2 ceTag-76 ceT23D8.7 dmAGO1 ceALG-1 ceALG-2 hsAGO3 hsAGO1 hsAGO4 hsAGO2

Niraj H. Tolia and Leemor Joshua-Tor

RDE-1

Alla Grishok

5 ' 3 '

• D

C R

• pre!miRNA

miRNA

Victor Ambros

Lee et al., 1993

Gary Ruvkun

Reinhart et al., 2000 Pasquinelli et al., 2000

21 nt mature miRNA lin-4 let-7

Small RNAs take center stage!

David Baulcombe/Plants

Hamilton and Baulcombe, 1999

Tom Tuschl/Vertebrate cells

Elbashir et al., 2001

Zamore, Tuschl, Sharp and Bartel/Drosophila

Zamore et al., 2000

Greg Hannon

Hammond et al., 2000

Alla Grishok and Amy Pasquinelli

Grishok et al, 2001

ALG-1/ALG-2 WT dcr-1 alg

Long dsRNA Stimulii Initiators Effectors? Transposons Transgenes rde-1 ? alg-1/alg-2 Small RNAs miRNA genes dcr-1 mRNA turn-

• ver

Transcriptional Control, Other Functions? Translation control Greg Hannon

with Claire (Bernstein et al., 2001)

Argonaugte aka ‘The Cop’ Who is the RNAi cop in

• C. elegans?

A model for mRNA slicing

Ji-Joon Song et al., 2004

L 4 y

• u

n g a d u l t g r a v i d a d u l t no RNAi Darryl Conte and Alla Grishok pos-1 mRNA

Burst of target mRNA expression before silencing

pos-1(RNAi) pos-1 mRNA

Only antisense siRNAs accumulate. Requires a target mRNA, RDE-1, RDE-4, DCR-1 and RDE-9

homogenize

Western Blot Pedro Batista

Affinity Matrix

m7G AAAAAA

DRH-1/2 m7G

mRNA Cleavage dsRNA trigger cleavage - primary siRNAs production

“RDE1:RISC complex”

m7G A A A A A A

RdRP Recruitment dsRNA synthesis Dicer cleavage secondary siRNAs production

Recycling of anti-sense siRNA loaded RDE1:RISC

3‘UTR

RdRP

RDE-1 RDE-1 RDE-1 RDE-1 DCR-1 DCR-1

RDE-4

RDE-1

RDE-1 Homolog?

RDE-1/siRNA complex Very low abundance

RDE-1 Homolog?

ceRDE-1 ceZK1248.7 ceF58G1.1 cePPW-2 ceR06C7.1 ceF55A12.1 ceY49F6A.1 ceR04A9.2 ceT22H9.3 ceC16C10.3 ceM03D4.6 ceK12B6.1 cePPW-1 ceF56A6.1 ceF20D12.1 ceC04F12.1 ceR09A1.1 ceD2030.6 ceTAG-292 hsHILI hsHIWI2 hsPIWIL3 hsHIWI dmAUB dmPIWI dmAGO2 spAGO1 atAGO5 atZLL/PNH atAGO1 atAGO6 atAGO4 atAaGO9 atAGO7 atAGO2 atAGO3 ceT22B3.2 ceTag-76 ceT23D8.7 dmAGO1 ceALG-1 ceALG-2 hsAGO3 hsAGO1 hsAGO4 hsAGO2

Niraj H. Tolia and Leemor Joshua-Tor

ppw-2 (I) C18 unc-29 (I) F58 (II) dpy-10 C06 (II) C04 (I) F56 (I) K12 (V) M03 (IV) unc-29 C18 (I) X X X X F20/nT1(gfp) (IV) +/nT1(gfp) (V) C18 F56/+ (I) C06 F58/+ (II) +/nT1(gfp) (IV) +/nT1(gfp) (V) C18 F56(I); C06 F58(II); M03(IV) K12(V) X X X F56 ppw-2 C18 C04 (I); unc-32 (III) F58 F56 ppw-2 C18 C04 (I) F58 (II) F56 + + + / + ppw-2 C18 C04 (I); unc-32 (III) hT2(gfp): him-8 (IV) ppw-2 C18 C04 (I); unc-32 (III) F56 ppw-2 C18 (I) + / + + C04; unc-32 hT2(gfp) (I) hT2(gfp) (III) ppw-2 C18 (I) unc-32 (III) ppw-2 C18 (I) dpy-5 (I); rol-6 (II); unc-32 (III) + ppw-2 +/ +C18 unc-29 (I) hT2(gfp)/ego-1 rde-9 (I); hT2/+; him-8 (IV) X X X X X X X NT1(gfp)/dis-3 F56 C18 F56 C18 (I) NT1(gfp)/+ (IV) NT1(gfp) (V) M03 (IV) K12 (V) F56 C18 (I) M03 (IV) K12 (V) X X C18 F56 (I) C06 F58 (II) hT2(gfp)/rde-9 (I); him-8(IV) C06 F58 (II) X hT2(gfp)/+ (I); C06 F58 (II); him-8/+ (IV) X C18 F56 (I)

Sextuple Argonaute Mutant

F56, ppw-2, C18, C04 (I); F58 (II);C06, M03 (IV), K12 V

Octuple

Erbay Yigit

Loss of Multiple Argonautes Leads to RNAi deficiency unc-22 RNAi

Paralyzed Mobile Twitcher Non Twitcher

paralyzed mobile twitcher non-twitcher 25 50 75 100 Genotype % Affected Animals

severity

W T C 6 M 3 K 1 2 F 5 6 F 5 8 C 1 8 F 5 8 s e x t u p l e C 1 8

myo-3::rde-1::HA

+++

• myo-3::gfp::f56
• +++

myo-3::ppw-1

• +++

myo-3::gfp::k12

• +++

rde-1 (ne300) sextuple Argonaute mutant Expression Vector Over-expression of individual Argonautes can Rescue the Sextuple Argonaute Mutant myo-3::prg-1

• myo-3::alg-1

ND

GFP Western unc-22 siRNA GFP::K12B6.1 Wild Type SAM GFP::F56A6.1 Wild Type SAM GFP::F56A6.1 GFP::K12B6.1 OP50 unc-22

Secondary AGOs Are Limiting for RNAi

W i l d T y p e S A M G F P : : F 5 6 A 6 . 1 G F P : : K 1 2 B 6 . 1 R D E 1

• /
• unc-22 siRNA

Erbay Yigit and Pedro Batista

Blot for small RNAs

m7G AAAAAA

DRH-1/2 m7G

mRNA Cleavage dsRNA trigger cleavage - primary siRNAs production “RDE1:RISC complex”

m7G AAAAAA

RdRP Recruitment dsRNA synthesis Dicer cleavage secondary siRNAs production

Recycling of anti-sense siRNA loaded RDE1:RISC 3‘UTR RdRP

RDE-1 RDE-1 RDE-1 RDE-1 DCR-1 DCR-1

RDE-4

RDE-1

Heterochromatin formation and Transcriptional Gene Silencing mRNA Degradation

(P-Body Recruitment?) F58 C06 K12 M03 PPW-1 F56 C16 PPW-2 ?

AAAAAA m7G

?

? Transponson & Transgene Silencing ?

Long dsRNA Triggers Initiators Effectors? Transposons Transgenes rde-1 ? alg-1/alg-2 Small RNAs miRNA genes dcr-1 mRNA turn-

• ver

Transcriptional Control, Other Functions? Translation control Long dsRNA Triggers Initiators Effectors Transposons Transgenes rde-1 ? alg-1/alg-2 miRNA genes mRNA turn-

• ver

Transcriptional Control, Other Functions? Translation control dcr-1 dcr-1 rde-1 homologs alg-1/alg-2

Chromatin: the physiological form of the genome

• T. J.

Richmond

Nucleosomes: the building blocks

• f Chromatin

Regulatory Modifications

Maintenance of silencing and gene-expression levels through cell division Inheritance? Reprogramming? Dosage compensation?

A Chromatin-RNA Feedback Loop “Active” “Silent” pol II mRNA

AGO

AGO

AGO

dsRNA pol ? pol ? siRNA

surveillance

Histone and tubulin GFP transgenes kindly provided by Geraldine Seydoux

Wildtype csr-1 mutant Julie Claycomb Ka Ming Pang

N2 n e 4 1 9 7 n e 4 2 5 4 n e 4 2 5 3 N 2 dcr-1 22 nt 20 nt 5S tRNA{ 5.8S

An abundant DCR-independent species of endo-siRNA Weifeng Gu

Nova Science Now Eric Lander

THANK YOU! Andy! University of Massachusetts Howard Hughes Medical Institute National Institutes of Health, USA Pew Scholars Program March of Dimes American Cancer Association . . . .