Science - USA (2020-03-13)

likely correspond to the RNA-recognition motif
and protein interaction domain of Orb2 (Fig.
4C, gray density). We could identify densities
that correspond to peptide-group oxygen atoms,
ordered solvent molecules in each interface, and
alternative conformations of histidine side
chains(fig.S10,AtoC).Consequently,we
could build and stereochemically refine an
atomic model de novo. The resulting model of
the amyloid core comprised 31 amino acid
residues from the glutamine-rich prion-like
domain of Orb2 (Fig. 4, D and E). Orb2 pro-
tein has two isoforms in the fly brain ( 10 )that

share the prion-like domain but differ in their

N-terminal extension (Fig. 4F). The low-

abundant Orb2A seeds aggregation of preva-

lent Orb2B, which can subsequently seed Orb2B

protein ( 10 ). Of the protein in our sample, 97.5%

is Orb2B (fig. S1C), and the protofilament core

structure extends from residues 176 to 206 of

Orb2B (Fig. 4, D and E).

The protofilament core adopts a simple

hairpin-like fold, composed of twob-strands,

b1(residues176to186)andb2 (residues 197 to

206), with a wide turn (residues 187 to 196) in

between (Fig. 4G). Perpendicular to the helical

axis, a difference in height of 6 Å between the

highest point in the turn and the lowest point

in the tip of theb2 allows an ordered hydrogen-

bonding network of theb-stranded regions

(Fig. 4H). The cross-bpacking of the two

b-strands is made of a tight interdigitation

of glutamine residues, Q179, Q181, Q183, and

Q185 fromb1 and Q200, Q202, and Q204

fromb2 (Fig. 4I). A single leucine, L198, sep-

arates this block of seven glutamines from a

clusteroffourmoreinteriorglutamineresi-

dues in the hairpin turn: Q187, Q190, Q193,

and Q196 (Fig. 4E). Next to H189, there is an

Hervaset al.,Science 367 , 1230–1234 (2020) 13 March 2020 3of5

Fig. 3. Biochemical activity of

Orb2 monomer, oligomer, and

filament isolated from adult head.

(A) Schematic representation of

the biochemical activity of Orb2.

CG13928 binds to Orb2 monomer

and recruit translation repression

complex (indicated in yellow).

CG4612 binds to aggregated Orb2

and recruit translation promoting

complex (indicated in green).

(B) Negative-stain EM of nanogold-

labeled 3′UTR of Tequila mRNA

bound to Orb2 filaments. Black

dots indicated with white arrows

are nanogold particle (~2 nm) attached

to target mRNA. (C) Immuno-EM

of CG4612 bound to Orb2 filaments.

Black dots indicated with black

arrows are gold particles (~6 nm)

attached to CG4612 protein. (D)EM

of Orb2 filaments bound to nanogold-

labeled 3′UTR of Tequila mRNA

and CG4612 protein. The larger

gold particle (~6 nm, black arrows)

represents CG4612 protein, and

the smaller gold particle (~2 nm,

white arrows) represents the mRNA.

(E) Translation of Orb2-target

mRNA in presence of different Orb2

species, obtained from embryo,

adult fly head, or seeding reaction.

Purified Orb2 monomer represses

translation, while Orb2 oligomer

and filament enhance translation. Bovine

serum albumin (BSA) was used as

a control. ***P= 0.0003, ****P<

0.0001; Student’sttest; two tailed.

Data are expressed as mean ± SEM.

AAA AAAAAAA^ AAA^

A A A AAAAA A

A^ AAA AAAAAA

Au

Mono-amino

nanogold

45 min RT

AAAAA

113 bp Tequila

3’UTR mRNA

CGUUGU M2P

UUUUGU Wt

Au-labelled-mRNA

BC

500 Å 500 Å 500 Å 500 Å

gold-labelled anti-CG4612

45 min RT

Au-labelled-

mRNA

500 Å

CG4612 CG4612Δ

Te q 5 ’ Teq 3’

Firefly

60 min RT

AT P

aa Mix

FF/RL

translation extractΔorb2 ratio

SV40 5’ SV40 3’

Renila

E

seeded

D

10x wash

+ Au +

mRNA-binding protein-binding

mRNA and protein-binding

A A

A

Orb2

Ribosomemonomer

PolyA tail

CG13928

A A

A

A A A

Ribosome filamentOrb2

PolyA tail

Translation

repression

Translation

activation

A

100 Å 100 Å

100 Å

Wt mRNA-Orb2 filament M2P mRNA-Orb2 filament CG4612-Orb2 filament CG4612Δ-Orb2 filament

Wt mRNA-CG4612-Orb2 filament M2P-CG4612Δ-Orb2 filament

CG4612

A

500

400

300

200

100

0

120

80

60

40

20

0

100

***

****

BSA

seeded seeded

BSA

Repression

complex CGG Activationcomplex

Rib Rib RibRiRiRib

A (^) A
A A
G46
(^) AA
46
(^) A AAAAAAAAA A AAAA A A AAAAAA
C CCCC
A A
A
A AA
A AAA
....
.
.
....
..
% Normalized FF/RL
or
or
Au
30 min RT, 30 min 4°C
CG4612 CG4612Δ

• 
  
  
  
  • gold-labelled
    anti-CG4612
  
  
  
  


• 
  
  
  
  • 10x wash
    M2P
    Wt
    M2P
    Wt
  
  
  
  


• 
  

  RESEARCH | REPORT