Download as PDF, PPTX
![T [°C]
time
33
20
35
[A1-FL] (μM)
50 100 150 200 250 300
Temperature(⁰C)
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
m7Gppp
AAA
m7Gppp
AAA
m7Gppp
AAA
m7Gppp
AAA
m7Gppp
AAA
RNA granule
m7Gppp
AAA
m7Gppp
AAA
m7Gppp
AAA
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](https://image.slidesharecdn.com/tayloralzforum-151102152031-lva1-app6891/85/Paul-Taylor-20-320.jpg)
![Persistent Granule Assembly Promotes Amyloid
Formation
m7Gppp
AAA
m7Gppp
AAA
m7Gppp
AAA
m7Gppp
AAA
Individual mRNPs
(one phase)
AAA
m7Gppp
RNA Granule
(two phases)
Pathological
inclusions
Reversible
phase
separation
AAA
m7Gppp
m7Gppp
AAA
m7Gppp
AAA
m7Gppp
AAA
m7Gppp
AAA
Amyloid
formation
AAA AAA
AAA AAA
m7Gppp m7Gppp
m7Gpppm7Gppp
m7Gppp
AAA
m7Gppp
AAA
m7Gppp
AAA
m7Gppp
AAA
[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](https://image.slidesharecdn.com/tayloralzforum-151102152031-lva1-app6891/85/Paul-Taylor-27-320.jpg)
Uploaded byAlzforum
Paul Taylor
The document discusses dynamic RNA-protein assemblies in neurological diseases, focusing on multisystem proteinopathy and mutations in various RNA-binding proteins like hnrnpa1 and hnrnpa2b1. It explores the clinical pleiotropy in patients with identical VCP mutations and the implications of these mutations on age-related degenerative diseases, highlighting the relationships among amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other related conditions. Additionally, it emphasizes the disturbance in RNA granule dynamics in these diseases, driven by factors such as phase separation and fibrillization tendencies of mutant proteins.
More Related Content
Paul Taylor
- 1. J. Paul Taylor,MD, PhD St. Jude Children’s Research Hospital Howard Hughes Medical Institute Dynamic RNA-protein assemblies in neurological disease
- 2. 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?
- 3. Family 1 Family3Family 2 Family 4 Family 5 Family 6 Family 7 Family 8 Family 9 Multisystem Proteinopathy Families Benatar et al. Neurology 2013
- 4. Mutations in hnRNPA2B1and hnRNPA1 cause MSP hnRNP A2B1 12 aa D290V D302V A2 ( 95%) B1 ( 5%) isoform LCS LCS MS9MS9 D262V hnRNP A1 52 aa a (95%) b ( 5%) isoform LCS LCS MS9MS9 D262V Kim et al., Nature 2013
- 5. Pinkus et al.,Neuromuscular diseases 2014
- 6. Gene Functional ClassMSP ALS FTD IBM Paget’s Protein present in pathology VCP Ubiquitin-dependent segregase ✔ ✔ ✔ ✔ ✔ yes p62/SQSTM1 Ubiquitin-dependent autophagy ✔ ✔ ✔ ✔ ✔ yes Optineurin Ubiquitin-dependent autophagy ✔ ✔ ✔ ✔ yes Ubiquilin2 Ubiquitin-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
- 7. Mutations in thesesame genes are found in sporadic diseas 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 FTD TDP43 FUS p62/SQSTM1 UBQLN2 VCP
- 8. 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) MS9 hnRNP A2B1 D302V (MSP) MS9 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
- 9. PrLDs contain a“steric zipper” motif that promotes fibrillization Core PrLD hnRNPA1 RRM1 RRM2 Glycine-Rich Domain M9 NLS 12 92 105 181 186 289268 320 GGYGGSGDGYNGFGNDGSNFGGGGSYNDFGNYNNQSSN 233 272 hnRNPA2 RRM1 RRM2 Glycine-Rich Domain M9 NLS 929 100 179181 296 319 341 Core PrLD NQGGGYGGGYDNYGGGNYGSGNYNDFGNYNQQPSNYGP 266 303 Zipper DB predictions
- 10. hnRNPA2B1 and hnRNPA1fibril assembly accelerated by disease mutations USup35 ORD M domain C domain PrLD ORD M domain C domain 1 40 114 254 685 S V Time (h) G inpellet(%) Time (h) H I J K M O Q inpellet(%) Time (h) WT D290V Δ287-292 WT D262V Δ259-264 WTD290VWTD262V 0h 4h 18h 0h 4h 12h μm μm inpellet(%) Time (h) 0 20 40 60 0 1 2 3 D290Vinpellet(%) Time (h) 0 10 20 30 0 1 2 3 4 40 50 Δ287-292inpellet(%) Time (h) 0 10 20 30 0 1 2 3 40 hnRNPA2inpellet(%) Time (h) 0 10 20 30 0 1 2 3 Sup35 Sup35 USup35 ORD M domain C domain PrLD ORD M domain C domain 1 40 114 254 685 S V Time (h) G inpellet(%) Time (h) H I J K M O Q inpellet(%) Time (h) WT D290V Δ287-292 WT D262V Δ259-264 WTD290VWTD262V 0h 4h 18h 0h 4h 12h μm μm inpellet(%) Time (h) 0 20 40 60 0 1 2 3 4 D290Vinpellet(%) Time (h) 0 10 20 30 0 1 2 3 4 40 50 5 6 Δ287-292inpellet(%) Time (h) 0 10 20 30 0 1 2 3 4 40 hnRNPA2inpellet(%) Time (h) 0 10 20 30 0 1 2 3 4 Sup35 Sup35 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
- 11. hnRNP incorporation intostress granules is enhanced by mutations hnRNPA2 D290V hnRNPA1 D262V Kim et al., Nature 2013
- 12.
- 13. Purified hnRNPA1 showstemperature-sensitive reversible turbidity 4oC 15 sec 25oC 15 sec 4oC 15 sec 25oC Albumin hnRNPA1
- 14. Purified Albumin in solution PurifiedhnRNPA1 in 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
- 15. Phase transition mediatedby LCD independent and distinct from fibrillization Molliex, Cell 2015
- 16. hnRNP protein dropletsexhibit liquid-like behavior time lapse time lapse Molliex, Cell 2015 Oregon Green-labeled droplets of purified hnRNPA1 GFP-tagged stress granules in cells
- 17. hnRNPs droplets arehighly 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
- 18. Time (ms)Relativeintensity RNA granulesare highly dynamic FRAP of GFP-G3BP Molliex, Cell 2015 Fluorescence recovery on a time scale of seconds
- 19. “Prion-like” low complexitydomain is sufficient to drive stress granule assembly Molliex, Cell 2015
- 20. T [°C] time 33 20 35 [A1-FL] (μM) 50100 150 200 250 300 Temperature(⁰C) 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 m7Gppp AAA m7Gppp AAA m7Gppp AAA m7Gppp AAA m7Gppp AAA RNA granule m7Gppp AAA m7Gppp AAA m7Gppp AAA 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
- 21. Genetics and cellbiology 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?
- 22. hnRNPA1 WT 5 µm hnRNPA1 D262V 5 µ m Merge 5 µ m 0 5 10 15 20 25 30 0 50 100 150 200 250 Temperature(⁰C) hnRNPA1 (μM) Diseases mutation in hnRNPA1 doesn’t impact phase separation hnRNPA1 WT and D262V are miscible in droplets D262V RRM1 RRM 2 LCS D262V RRM1 RRM 2 LCS hnRNPA1 hnRNPA1-D262V D262V Molliex, Cell 2015
- 23. Phase separation-dependent fibrillization Imagingthe slide surface after each cycle A1 D262V 110 uM 2d cycle 3rd cycle 4th cyclePre-cycling
- 24. Molliex, Cell 2015 •Imaging at the slide surface • hnRNPA1 D262V 140 μM + Thio-Flavin T 50 μM Pre-phase separation Held in 2-phase regime Phase separation-dependent fibrillization
- 25. Phase separation drivesfibrillization Imaging floating droplets Imaging the coverslip surface Wild type hnRNPA1 Mutant hnRNPA1
- 26. RRM RRM Lowcomplexity sequence Multiple adhesive domains Maturation (proto-fibrillization)Fibrillization
- 27. Persistent Granule AssemblyPromotes Amyloid Formation m7Gppp AAA m7Gppp AAA m7Gppp AAA m7Gppp AAA Individual mRNPs (one phase) AAA m7Gppp RNA Granule (two phases) Pathological inclusions Reversible phase separation AAA m7Gppp m7Gppp AAA m7Gppp AAA m7Gppp AAA m7Gppp AAA Amyloid formation AAA AAA AAA AAA m7Gppp m7Gppp m7Gpppm7Gppp m7Gppp AAA m7Gppp AAA m7Gppp AAA m7Gppp AAA [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
- 28. DAPI VCPwt eIF3B mergeTDP-43 VCPA232E VCP VCPR155H VCP mutationsdrive spontaneous SGs that contain disease-related RBPs Buchan et al, Cell 2013
- 29. time RNAGranuleFormation Assembly > Disassembly RNAgranule 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
- 30. RNA Granule mRNP mRNPs Nuclear exportand exchange of RBPs Piecemeal degradation by autophagy RNA Granule Assembly: • Impacted by mutations in RNA-binding proteins • TDP-43, FUS, hnRNPA1 Nucleus Proteasomal degradation Polysome m7Gppp AAA m7Gppp AAA m7Gppp AAA m7Gppp AAA m7Gppp AAA m7Gppp AAA 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
- 31. Collaborators: Tanja Mittag Jihun Lee TaylorLab: Amandine Molliex Hong Joo Kim Maura Coughlin Anderson Kanagaraj St. Jude Imaging Resource: Jamshid Temirov