RNAi and Development in C. elegans Craig C. Mello Stockholm December 10, 2006 UMass
Caenorhabditis elegans Bob Goldstei !
Sydney Brenner David Hirsh Bob Horvitz John Sulston Dan Stinchcomb Victor Ambros Jim Priess
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?
RNA-directed inheritance? Weismann’s theory (ala Ernst Mayr/& post RNAi). (1)There is a special particle (biophore) for each trait. ( with siRNAs ) some s. (2)These particles can grow and multiply independent of siRNAs cell division. (3)Both nucleus and cytoplasm consist of these biophores. siRNAs. contain (4)A given biophore may be represented by many replicas siRNA 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 siRNAs division). “As we now know, postulates (2) and (5) are wrong and NOT WRONG. are responsible for the fact that Weismann was not able RNA may play a role in inheritance and evolution. to arrive at a correct theory of inheritance.”
cbs news explains RNAi
Nova Science Now Robert Krulwich Eric Lander
dsRNA targetting RNA injection essential maternal genes carrier carrier unaffected rde/mut(+) P0 oocytes oocytes embryos Carriers F1 carriers Potent rde/mut (-) F1 uninjected X F1 Sequence-specific rde/mut (-) F2 F2 affected dead eggs F3 dead eggs Heritable Systemic Amplified!
RNAi Catalytic model of RNAi Injected dsRNA Foreign dsRNA Recognition Degradation Target tissue Transport into Transport into the the cells cells Transport pre-mRNA RNA-dependent guided RNA-degradation Silencing Accumulation Accumulation of of RNAi RNAi active active molecules molecules in in the the target target tissue tissue Amplification and Inheritance
Hiroaki’s screen for RNAi deficient mutants (1998-1999) Mutagenize P0 Feed dsRNA F2 (Lisa Timmons) RNAi-deficient wt RNAi induced viable progeny dead eggs Hiroaki Tabara
Analysis of Hiroaki’s RNAi Deficient Mutants # alleles Other Phenotypes rde-1 6 none mutator rde-2 3 ! sterile/him/mutator rde-3 3 sterile/him/mutator rde-4 2 none mutator rde-5 1 sterile/him/mutator mutator rde-6 2 sterile/him/mutator mutator mut-7 1 sterile/him/mutator
RNAi Transposons Transgenes What about RDE-1? Hypothesis: Related silencing pathways with distinct triggering mechanisms
Argonaute protein RDE-1 1020aa PAZ PIWI 618 985 334 443 C. elegans Drosophila Arabidopsis Neurospora Argonaute protein RDE-1 Aubergine/Sting AGO1 QDE-2 RDE-1 1020aa PAZ PIWI 618 985 334 443 RNAi Development and Development Quelling Epigenetic silencing Gene silencing (transgene silencing (Tabara et al., 1999) (Schmidt et al., 1999 (Fagard et al., 2000 Cogoni and Macino, 2000 Wilson et al., 1996) Bohmert et al., 1998) RDE-1 Cloning reveals links to development and Hiroaki Tabara gene silencing in insects, plants and fungi!
atAGO1 atAGO4 Niraj H. Tolia and Leemor Joshua-Tor atAaGO9 atZLL/PNH atAGO6 atAGO7 atAGO2 atAGO5 atAGO3 dmPIWI dmAUB hsHIWI spAGO1 ceT22B3.2 ceTag-76 dmAGO2 ceALG-1 hsPIWIL3 ceT23D8.7 dmAGO1 ceALG-2 hsHIWI2 hsHILI hsAGO3 RDE-1 ceTAG-292 ceRDE-1 ceR09A1.1 hsAGO1 ceD2030.6 ceC04F12.1 ceF20D12.1 hsAGO4 Alla Grishok ceF56A6.1 ceZK1248.7 cePPW-1 ceK12B6.1 ceM03D4.6 hsAGO2 ceY49F6A.1 ceC16C10.3 ceR04A9.2 ceF58G1.1 ceT22H9.3 cePPW-2 ceF55A12.1 ceR06C7.1
��� �������� ������������ ������� ���� Small RNAs take center stage! WT dcr-1 alg lin-4 let-7 5 ' 3 ' David Baulcombe/Plants pre ! miRNA Hamilton and Baulcombe, 1999 Tom Tuschl/Vertebrate cells D C R ALG-1/ALG-2 Elbashir et al., 2001 Zamore, Tuschl, Sharp and Victor Ambros Lee et al., 1993 Bartel/Drosophila Zamore et al., 2000 Gary Ruvkun Reinhart et al., 2000 Greg Hannon Pasquinelli et al., 2000 Hammond et al., 2000 Alla Grishok 21 nt and Amy Pasquinelli miRNA mature miRNA Grishok et al, 2001
Long Transposons miRNA Stimulii dsRNA Transgenes genes rde-1 ? alg-1/alg-2 Initiators dcr-1 Small RNAs Effectors? Greg Hannon Translation mRNA turn- Transcriptional with Claire control over (Bernstein et al., 2001) Control, Other Functions?
A model for mRNA slicing Argonaugte Who is the aka ‘The Cop’ RNAi cop in C. elegans? Ji-Joon Song et al., 2004
t t l l u u d d a a g d n i v u a 4 o r L g y pos-1 mRNA no RNAi pos-1 mRNA pos-1(RNAi) Darryl Conte and Alla Grishok Burst of target mRNA expression before silencing
Only antisense siRNAs accumulate. Requires a target mRNA, RDE-1, RDE-4, DCR-1 and RDE-9
homogenize Affinity Matrix Western Blot Pedro Batista
DRH-1/2 dsRNA trigger cleavage - DCR-1 primary siRNAs production RDE-1 RDE-4 RDE-1/siRNA complex Very low abundance RDE-1 RDE-1 “RDE1:RISC complex” RDE-1 3‘UTR Recycling of m7G AAAAAA anti-sense siRNA loaded RDE1:RISC mRNA Cleavage RDE-1 RdRP Recruitment dsRNA synthesis A A A A A A m7G Dicer cleavage DCR-1 RdRP m7G secondary siRNAs production RDE-1 Homolog? RDE-1 Homolog?
Niraj H. Tolia and Leemor Joshua-Tor atAGO1 atAGO4 atAaGO9 atZLL/PNH atAGO6 atAGO7 atAGO2 atAGO5 atAGO3 dmPIWI dmAUB hsHIWI spAGO1 ceT22B3.2 ceTag-76 dmAGO2 ceALG-1 hsPIWIL3 ceT23D8.7 dmAGO1 ceALG-2 hsHIWI2 hsHILI hsAGO3 ceTAG-292 ceRDE-1 ceR09A1.1 hsAGO1 ceD2030.6 ceC04F12.1 ceF20D12.1 hsAGO4 ceF56A6.1 ceZK1248.7 cePPW-1 ceK12B6.1 ceM03D4.6 hsAGO2 ceY49F6A.1 ceC16C10.3 ceR04A9.2 ceF58G1.1 ceT22H9.3 cePPW-2 ceF55A12.1 ceR06C7.1
X dpy-10 C06 (II) F58 (II) X hT2(gfp)/rde-9 (I); him-8(IV) C06 F58 (II) Sextuple Argonaute Mutant hT2(gfp)/+ (I); C06 F58 (II); him-8/+ (IV) X C18 F56 (I) X F20/nT1(gfp) (IV) +/nT1(gfp) (V) C18 F56 (I) C06 F58 (II) C18 F56/+ (I) C06 F58/+ (II) +/nT1(gfp) (IV) +/nT1(gfp) (V) Octuple X C18 F56(I); C06 F58(II); M03(IV) K12(V) F56 C18 (I) M03 (IV) K12 (V) F56, ppw-2, C18, C04 (I); F58 (II);C06, M03 (IV), K12 V X X F56 C18 (I) NT1(gfp)/+ (IV) NT1(gfp) (V) M03 (IV) K12 (V) F56 ppw-2 C18 C04 (I) F58 (II) X K12 (V) M03 (IV) X NT1(gfp)/dis-3 F56 C18 X F56 ppw-2 C18 C04 (I); unc-32 (III) F58 unc-29 C18 (I) X F56 (I) X F56 + + + / + ppw-2 C18 C04 (I); unc-32 (III) hT2(gfp): him-8 (IV) X ppw-2 C18 C04 (I); unc-32 (III) F56 X ppw-2 C18 (I) + / + + C04; unc-32 hT2(gfp) (I) hT2(gfp) (III) X ppw-2 C18 (I) unc-32 (III) C04 (I) Erbay Yigit X ppw-2 C18 (I) dpy-5 (I); rol-6 (II); unc-32 (III) X + ppw-2 +/ +C18 unc-29 (I) hT2(gfp)/ego-1 rde-9 (I); hT2/+; him-8 (IV) X C18 unc-29 (I) ppw-2 (I)
Loss of Multiple Argonautes Leads to RNAi deficiency unc-22 RNAi severity 100 paralyzed Paralyzed % Affected Animals mobile twitcher Mobile Twitcher 75 non-twitcher Non Twitcher 50 25 0 C W C M K F F C s e 5 5 1 0 1 1 0 8 2 8 x T 6 8 6 3 t F u 5 p 8 l e Genotype
Over-expression of individual Argonautes can Rescue the Sextuple Argonaute Mutant sextuple Argonaute Expression Vector rde-1 (ne300) mutant +++ - myo-3::rde-1::HA - +++ myo-3::gfp::f56 - +++ myo-3::ppw-1 - +++ myo-3::gfp::k12 - - myo-3::prg-1 ND - myo-3::alg-1
Secondary AGOs Are Limiting for RNAi 1 1 . . 6 6 A B e 2 - 6 p / 1 5 - y K T F 1 : : M E d : : P P D l i A F F W G G R S Blot for small RNAs unc-22 siRNA OP50 unc-22 GFP::K12B6.1 GFP::K12B6.1 GFP::F56A6.1 GFP::F56A6.1 Wild Type Wild Type SAM SAM GFP Western unc-22 siRNA Erbay Yigit and Pedro Batista
DRH-1/2 dsRNA trigger cleavage - DCR-1 primary siRNAs production RDE-1 RDE-4 RDE-1 RDE-1 “RDE1:RISC complex” RDE-1 3‘UTR Recycling of m7G AAAAAA anti-sense siRNA loaded RDE1:RISC mRNA Cleavage RDE-1 RdRP Recruitment dsRNA synthesis AAAAAA Transponson m7G & Transgene Dicer cleavage DCR-1 ? RdRP m7G secondary siRNAs Silencing production K12 PPW-2 F56 F58 C16 PPW-1 C06 ? M03 mRNA Degradation (P-Body Recruitment?) Heterochromatin formation and ? ? AAAAAA m7G Transcriptional Gene Silencing
Long Long Transposons Transposons miRNA miRNA Triggers Triggers dsRNA dsRNA Transgenes Transgenes genes genes rde-1 rde-1 ? ? alg-1/alg-2 alg-1/alg-2 dcr-1 dcr-1 Initiators Initiators dcr-1 alg-1/alg-2 rde-1 homologs Small RNAs Effectors Effectors? Translation Translation mRNA turn- mRNA turn- Transcriptional Transcriptional control control over over Control, Control, Other Functions? Other Functions?
Chromatin: the physiological form of the genome Nucleosomes: the building blocks T. J. of Chromatin Richmond
Regulatory Modifications
A Chromatin-RNA Feedback Loop “Active” “Silent” mRNA surveillance AGO pol ? pol II pol ? AGO dsRNA Maintenance of silencing and gene-expression levels AGO through cell division Inheritance? siRNA Reprogramming? Dosage compensation?
Wildtype Julie Claycomb Ka Ming Pang csr-1 mutant Histone and tubulin GFP transgenes kindly provided by Geraldine Seydoux
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