paul taylor
TRANSCRIPT
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