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Moving Pieces in a RISC Loading Complex Hong-Wei Wang (Yale) David Taylor (Yale) Bunpote Siridechadilok* Jennifer Doudna Ian MacRae (Scripps) Cameron Noland Enbo Ma Karin Felderer *(NCGEB, Thailand)

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Page 1: Moving Pieces in a RISC Loading Complex

Moving Pieces in a RISC Loading Complex

Hong-Wei Wang (Yale)David Taylor (Yale)

Bunpote Siridechadilok*Jennifer Doudna

Ian MacRae (Scripps)Cameron Noland

Enbo MaKarin Felderer

*(NCGEB, Thailand)

Page 2: Moving Pieces in a RISC Loading Complex

© The Nobel Committee for Physiology or Medicine Illustration: Annika Rohl

RNA interference (RNAi)

Page 3: Moving Pieces in a RISC Loading Complex

Gregory et al, Cell 2005

Dicer (~200 kDa): RNaseIII enzyme – dsRNA processingTRBP (~50 kDa): dsRNA binding protein – sensor for strand selectionArgonaute2 (Ago2, ~100 kDa): “slicer” – target mRNA cleavage

human RISC-Loading Complex (RLC)

Page 4: Moving Pieces in a RISC Loading Complex

Ago with guide siRNAYang et al., Nature 2008

Ago with guide siRNA & target RNAYang et al., Nature 2008

Crystal Structures of Argonaute

Ago with externally bound siRNAYuan et al., Structure 2006

First Ago StructureSong et al, Science 2004

Page 5: Moving Pieces in a RISC Loading Complex

Crystal structure of Dicer

MacRae et al, Science 2006 MacRae et al, CSHSQB 2006

Page 6: Moving Pieces in a RISC Loading Complex

55o

θ

φ

0

0.5

1

FSC

104 52 32 26 21 17 15 Å

EM single particle analysis of human Dicer

Page 7: Moving Pieces in a RISC Loading Complex

90o 90o 90o

90o

cap

base branch

platform

EM single particle reconstruction of human Dicer

Page 8: Moving Pieces in a RISC Loading Complex

WT dicer model reproj

ΔDEAD synthetic model reproj

ΔDEAD ref-free class average

Localization of DExH/D domain in human Dicer

Page 9: Moving Pieces in a RISC Loading Complex
Page 10: Moving Pieces in a RISC Loading Complex

Domain Docking

Page 11: Moving Pieces in a RISC Loading Complex

In vitro reconstitution of human RISC loading complex

MacRae IJ et al. PNAS 2008

Page 12: Moving Pieces in a RISC Loading Complex

90o 90ocap

base branch

platform

RLC Structure?Unstable RLC on EM grid

Kd: 0.2~1 µM Diluted conc. 30~50 nM

Page 13: Moving Pieces in a RISC Loading Complex

0

50

100

150

200

250

-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1

CCright-CCleft (scaled)

parti

cle

num

bers

I II III

I II III

Sorting of Heterogeneity by Supervised Classification

Page 14: Moving Pieces in a RISC Loading Complex

Dicer-Ago-TRBP

Dicer-Ago

III III IV

II’I’

TRBP stabilizes Ago-Dicer interaction

ML Volumes

Page 15: Moving Pieces in a RISC Loading Complex

Crosslinked human RISC loading complex

0 0.00

36

0.00

5

0.00

7

0.09

5

0.01

2

0.01

4

0.02

0.03

5

% Glutaraldehyde

Dicer

Ago2

TRBP

Page 16: Moving Pieces in a RISC Loading Complex

Category 1

Category 2

Category 3

Human RLC

Human Dicer + Ago2

Page 17: Moving Pieces in a RISC Loading Complex

Ago 2

DicerTRBP

Human RLC MODEL

Page 18: Moving Pieces in a RISC Loading Complex

Graphix-Purified RLC

Page 19: Moving Pieces in a RISC Loading Complex

90o 90o 90o

90o

90o 90o 90o

Graphix-Purified RLC

Page 20: Moving Pieces in a RISC Loading Complex

Ago 2

Dicer

TRBP

Ago 2

DicerTRBP

Model of RNA Loading

Page 21: Moving Pieces in a RISC Loading Complex

Hong-Wei Wang (Yale)David Taylor (Yale)

Bunpote Siridechadilok*Jennifer Doudna

Ian MacRae (Scripps)Cameron Noland

Enbo MaKarin Felderer

*(NCGEB, Thailand)

Page 22: Moving Pieces in a RISC Loading Complex
Page 23: Moving Pieces in a RISC Loading Complex
Page 24: Moving Pieces in a RISC Loading Complex

Figure S2

Page 25: Moving Pieces in a RISC Loading Complex

Figure S5


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