J. Paul Taylor, MD, PhD

St. Jude Children’s Research Hospital

Howard Hughes Medical Institute

Dynamic RNA-protein assemblies in

neurological disease

Multisystem Proteinopathy – a pleiotropic

syndrome

Frontotemporal dementia

ALS

Inclusion body Myopathy

Paget’s Disease of bone

Index case

Johnston et al. Neuron 2010

Watts et al. Nature Genetics 2004

R155H mutation in VCP

Frontotemporal dementia

ALS

Inclusion body Myopathy

Paget’s Disease of bone

Index case

R155H mutation in VCP

• What is the basis of clinical pleiotropy in patients with identical VCP mutations?

• What does this tell us about the etiologic relationship between these distinct age-related degenerative diseases?

• What is the basis of clinical pleiotropy in patients with identical VCP mutations?

• What does this tell us about the etiologic relationship between these distinct age-related degenerative diseases?

Family 1 Family 3Family 2

Family 4 Family 5

Family 6Family 7

Family 8

Family 9

Multisystem Proteinopathy Families

Benatar et al. Neurology 2013

Mutations in hnRNPA2B1 and hnRNPA1 cause MSP

hnRNP A2B1

12 aa

D290V

D302V

A2 ( 95%)

B1 ( 5%)

isoform

LCS

LCS

MS

9M

S9

D262V

hnRNP A1

52 aa

a (95%)

b ( 5%)

isoform

LCS

LCS

MS

9M

S9

D262V

Kim et al., Nature 2013

Pinkus et al., Neuromuscular diseases 2014

Gene Functional Class MSP ALS FTD IBM Paget’s

Protein

present in

pathology

VCPUbiquitin-dependent

segregase✔ ✔ ✔ ✔ ✔ yes

p62/SQSTM1Ubiquitin-dependent

autophagy✔ ✔ ✔ ✔ ✔ yes

OptineurinUbiquitin-dependent

autophagy✔ ✔ ✔ ✔ yes

Ubiquilin2Ubiquitin-dependent

autophagy✔ ✔ ✔ yes

TDP-43 RNA-binding protein ✔ ✔ yes

hnRNPA2B1 RNA-binding protein ✔ ✔ ✔ yes

hnRNPA1 RNA-binding protein ✔ ✔✔

yes

hnRNPDL RNA-binding protein ✔ yes

TIA-1 RNA-binding protein ✔ ✔ ✔ yes

The Molecular Genetics

of Multisystem Proteinopathy

Taylor, Neurology 2015

Mutations in these same genes are found in sporadic disease

ALS

hnRNPA1

TDP43

MATR3

FUS

p62/SQSTM1

UBQLN2

OPTN

VCP

IBM

hnRNPA2B1

MATR3

FUS

p62/SQSTM1

UBQLN2

VCP

Paget’s

hnRNPA1

hnRNPA2B1

p62/SQSTM1

UBQLN2

OPTN

VCP

FTDTDP43

FUS

p62/SQSTM1

UBQLN2

VCP

Disease mutations impact “low complexity sequence”

domains

80 %

53 Glycine

12 Asparagine

12 Tyrosine

9 Serine

7 Phenylalanine

5 Aspartic acid

4 Arginine

4 Proline

1 Glutamine

0 Threonine

0 Alanine

0 Methionine

0 Glutamic acid

0 Lysine

0 Cysteine

0 Histidine

0 Valine

0 Leucine

0 Isoleucine

0 Tryptophan

Inspired by Steve McKnight

TDP-43

hnRNP A1

D262V (fALS)

D262N (MSP)N267S (sALS)

MS

9

hnRNP A2B1

D302V (MSP)

MS

9

Q335Y (hIBM)

hnRNP DL

D378N (hIBM)D378H (hIBM)

Vieira et al. 2014

TIA-1

E384K (hIBM)

Kim et al. 2013

Kim et al. 2013

Klar et al. 2013

PrLDs contain a “steric zipper” motif

that promotes fibrillization

Core PrLD

hnRNPA1

RRM1 RRM2 Glycine-Rich DomainM9

NLS

12 92 105 181 186 289268 320

GGYGGSGDGYNGFGNDGSNFGGGGSYNDFGNYNNQSSN

233 272

hnRNPA2

RRM1 RRM2 Glycine-Rich DomainM9

NLS

929 100 179181 296 319 341

Core PrLD

NQGGGYGGGYDNYGGGNYGSGNYNDFGNYNQQPSNYGP

266 303

Zipper DB predictions

hnRNPA2B1 and hnRNPA1 fibril assembly accelerated

by disease mutations

T Gene Over-expression

A2 WT

A2 WT

A2 WT

A2 D290V

A2 D290V

A2 D290V

no

no

yes

yes

yes

yes

USup35

ORD M domain C domain

PrLD ORD M domain C domain

1 40 114 254 685S V

Figure 5 Kim et al.

A Δ287-292

Δ259-264B

C

D

V

Time (h)

Time (h)

V

E

F

G

100 nm

100 nm

V

V

in p

ellet (%

)

Time (h)

H

I

J

K L

M N

O P

Q R

in p

ellet (%

)

Time (h)

WTD290V

Δ287-292

WTD262V

Δ259-264

WT

D290V

WT

D262V

0h 4h 18h

0h 4h 12h

μm

μm

in p

ellet (%

)

Time (h)

in p

ellet (%

)

Time (h)

0

20

40

60

0 1 2 3 40

20

30

40

0 1 2 3 4

10

D29

0V

in

pellet (%

)

Time (h)

0

10

20

30

0 1 2 3 4

40

50

5 6

D2

62V

in p

ellet (%

)

Time (h)

0

10

2030

0 1 2

405060

70

Δ2

87

-292

in p

ellet (%

)

Time (h)

0

10

20

30

0 1 2 3 4

40

Δ259

-26

4 in p

ellet (%

)

Time (h)

0

10

20

30

0 1 2 3 4

40

hn

RN

PA

2 in

pellet (%

)

Time (h)

0

10

20

30

0 1 2 3 4

hn

RN

PA

1 in p

ellet (%

)

Time (h)

0

10

20

30

0 1 2 3 4

Sup35Sup35

1-

1+

2-

2+

3-

3+4-

4+

5-

5+

6-

6+

Donor

-

+

-

-

-

-

+

+

+

+

Cyto 1

Cyto 2

Cyto 3

Cyto 4

Cyto 5

T Gene Over-expression

A2 WT

A2 WT

A2 WT

A2 D290V

A2 D290V

A2 D290V

no

no

yes

yes

yes

yes

USup35

ORD M domain C domain

PrLD ORD M domain C domain

1 40 114 254 685S V

Figure 5 Kim et al.

A Δ287-292

Δ259-264B

C

D

V

Time (h)

Time (h)

V

E

F

G

100 nm

100 nm

V

V

in p

ellet (%

)

Time (h)

H

I

J

K L

M N

O P

Q R

in p

ellet (%

)

Time (h)

WTD290V

Δ287-292

WTD262V

Δ259-264

WT

D290V

WT

D262V

0h 4h 18h

0h 4h 12h

μm

μm

in p

ellet (%

)

Time (h)

in p

ellet (%

)

Time (h)

0

20

40

60

0 1 2 3 40

20

30

40

0 1 2 3 4

10

D29

0V

in

pellet (%

)

Time (h)

0

10

20

30

0 1 2 3 4

40

50

5 6

D2

62V

in p

ellet (%

)

Time (h)

0

10

2030

0 1 2

405060

70

Δ2

87

-292

in p

ellet (%

)

Time (h)

0

10

20

30

0 1 2 3 4

40

Δ259

-26

4 in p

ellet (%

)

Time (h)

0

10

20

30

0 1 2 3 4

40

hn

RN

PA

2 in

pellet (%

)

Time (h)

0

10

20

30

0 1 2 3 4

hn

RN

PA

1 in p

ellet (%

)

Time (h)

0

10

20

30

0 1 2 3 4

Sup35Sup35

1-

1+

2-

2+

3-

3+4-

4+

5-

5+

6-

6+

Donor

-

+

-

-

-

-

+

+

+

+

Cyto 1

Cyto 2

Cyto 3

Cyto 4

Cyto 5

hnRNPA2

hnRNPA1

• Full-length wild type hnRNPA2 and hnRNPA1 fibrillize after a lag phase

• Disease mutations greatly reduce the lag phase

• Deletion of the hexapeptide “steric zipper” eliminates fibrilization Kim et al., Nature 2013

hnRNP incorporation into stress granules is

enhanced by mutations

hnRNPA2 D290V

hnRNPA1 D262V

Kim et al., Nature 2013

D

D

Stress granules

Purified hnRNPA1 shows temperature-sensitive reversible turbidity

4oC 15 sec 25oC 15 sec 4oC 15 sec 25oC

Albumin hnRNPA1

Purified Albumin in solution

Purified hnRNPA1in solution

PrLDs allow hnRNPs to spontaneously assemble into liquid-like droplets

Purified hnRNPA1

Molliex, Cell 2015 : hnRNPA1 and TDP-43

Lin, Mol Cell 2015 : FUS, hnRNPA1 and other RBPs

Patel, Cell 2015 : FUS

Phase transition mediated by LCD independent and

distinct from fibrillization

Molliex, Cell 2015

hnRNP protein droplets exhibit liquid-like behavior

time lapse time lapseMolliex, Cell 2015

Oregon Green-labeled

droplets of purified hnRNPA1

GFP-tagged

stress granules

in cells

hnRNPs droplets are highly dynamic

Hydrogels show

no fluorescence

recovery from

sequential

bleaching even

after 16 minutes

Protein droplets

show fluorescence

recovery on a time

scale of seconds

hnRNPA1 Hydrogel

hnRNPA1 droplet

Molliex, Cell 2015

Time (ms)R

ela

tive inte

nsity

RNA granules are highly dynamic

FRAP of GFP-G3BP

Molliex, Cell 2015

Fluorescence recovery on a time scale of seconds

“Prion-like” low complexity domain is sufficient to drive stress

granule assembly

Molliex, Cell 2015

T [°C]

time

33

20

35

[A1-FL] (μM)

50 100 150 200 250 300

Tem

pera

ture

(⁰

C)

10

15

20

25

30

35

150 100 75

[Ficoll] (mg/ml)

hnRNPA1 is poised at the phase boundary as we

approach physiological conditions of temperature,

concentration, salt and intracellular molecular

crowding

Mapping the phase diagram of liquid-liquid

phase separation

Individual mRNPs

m7GpppAAA

m7GpppAAA

m7GpppAAA

m7GpppAAA

m7GpppAAA

RNA granule

m7GpppAAA

m7GpppAAA

m7GpppAAA

LLPS

Hypothesis: phase separation by LCD-containing

RBPs underlies the formation of RNA granules and

related RNA-protein assemblies as well as their

liquid properties

Molliex, Cell 2015

Genetics and cell biology point to a

disturbance in RNA granule

dynamics in ALS and related

diseases

Enigma

Patient pathology is dominated by

deposition of RNA-binding protein

inclusions

What is the relationship between hyperassembly of persistent

stress granules and deposition of RBP aggregates?

hnRNPA1 WT

5 µ m

hnRNPA1 D262V

5 µ m

Merge

5 µ m

0

5

10

15

20

25

30

0 50 100 150 200 250

Tem

pe

ratu

re (

⁰C)

hnRNPA1 (μM)

Diseases mutation in hnRNPA1 doesn’t

impact phase separation

hnRNPA1 WT and D262V are miscible in droplets

D262V

RRM1RRM

2LCS

D262V

RRM1RRM

2LCS

hnRNPA1

hnRNPA1-D262V

D262V

Molliex, Cell 2015

Phase separation-dependent fibrillization

Imaging the slide surface after each cycle

A1 D262V 110 uM

2d cycle 3rd

cycle

4th cyclePre-cycling

Molliex, Cell 2015

• Imaging at the slide surface

• hnRNPA1 D262V 140 μM + Thio-Flavin T 50μM

Pre-phase separation Held in 2-phase

regime

Phase separation-dependent fibrillization

Phase separation drives fibrillization

Imaging floating droplets

Imaging the coverslip surface

Wild type hnRNPA1 Mutant hnRNPA1

RRM RRM Low complexity sequence

Multiple adhesive domains

Maturation (proto-fibrillization)Fibrillization

Persistent Granule Assembly Promotes Amyloid

Formation

m7GpppAAA

m7GpppAAA

m7GpppAAA

m7GpppAAA

Individual mRNPs

(one phase)

AAA

m7Gppp

RNA Granule

(two phases)

Pathological

inclusions

Reversible

phase

separation

AAA

m7Gppp

m7GpppAAA

m7GpppAAA

m7GpppAAA

m7GpppAAA

Amyloid

formation

AAA AAA

AAA AAA

m7Gppp m7Gppp

m7Gpppm7Gppp

m7GpppAAA

m7GpppAAA

m7GpppAAA

m7GpppAAA

[hnRNP]low = low risk

of amyloid formation

[hnRNP]high = high risk

of amyloid formation

time

Insoluble residua

formed from the

most

amyloidogenic

constituents of

granules

DAPI

VC

Pw

t

eIF3B mergeTDP-43

VC

PA

23

2E

VCP

VC

P R

15

5H

VCP mutations drive spontaneous SGs that

contain disease-related RBPs

Buchan et al, Cell 2013

time

RN

A G

ran

ule

Fo

rmati

on

Assembly > Disassembly

RNA granule dynamics

Mutations or other factors

promote granule assembly

(e.g. PrLD mutations)

Mutations or other factors

impede granule disassembly

(e.g. VCP mutations)

Assembly < Disassembly

Assembly = Disassembly

RNA Granule Hyperassembly

RNA Granule

mRNP

mRNPs

Nuclear export and

exchange of RBPs

Piecemeal degradation

by autophagy

RNA Granule Assembly:

• Impacted by mutations in

RNA-binding proteins

• TDP-43, FUS, hnRNPA1Nucleus

Proteasomal

degradation

Polysome

m7GpppAAA

m7GpppAAA

m7GpppAAA

m7GpppAAA

m7GpppAAA

m7GpppAAA

RNA Granule Disassembly

and Clearance:

• Impacted by mutations in

disassembly factors and

catabolic pathways

• VCP, p62/SQSTM1,

UBQLN2

Integrated View of Disease Genetics and RNA Granule Dynamics

Collaborators:

Tanja Mittag

Jihun Lee

Taylor Lab:

Amandine Molliex

Hong Joo Kim

Maura Coughlin

Anderson KanagarajSt. Jude Imaging Resource:

Jamshid Temirov