Mechanism of karyopherin-β2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X)
Abstract
Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid–liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-β2 (Kapβ2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapβ2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapβ2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapβ2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapβ2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapβ2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.
- Authors:
-
- Univ. of Texas Southwestern Medical Center, Dallas, TX (United States)
- Ritsumeikan University, Shiga (Japan)
- Publication Date:
- Research Org.:
- Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institute of General Medical Sciences (NIGMS); Welch Foundation; Japan Society for the Promotion of Science (JSPS KAKENHI)
- OSTI Identifier:
- 1772764
- Grant/Contract Number:
- AC02-06CH11357; R01GM069909; I-1532; JP19K16060; JP19dm0307032
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Scientific Reports
- Additional Journal Information:
- Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 2045-2322
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; biochemistry; biophysics; diseases; molecular biology; structural biology
Citation Formats
Gonzalez, Abner, Mannen, Taro, Çağatay, Tolga, Fujiwara, Ayano, Matsumura, Hiroyoshi, Niesman, Ashley B., Brautigam, Chad A., Chook, Yuh Min, and Yoshizawa, Takuya. Mechanism of karyopherin-β2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X). United States: N. p., 2021.
Web. doi:10.1038/s41598-021-83196-y.
Gonzalez, Abner, Mannen, Taro, Çağatay, Tolga, Fujiwara, Ayano, Matsumura, Hiroyoshi, Niesman, Ashley B., Brautigam, Chad A., Chook, Yuh Min, & Yoshizawa, Takuya. Mechanism of karyopherin-β2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X). United States. https://doi.org/10.1038/s41598-021-83196-y
Gonzalez, Abner, Mannen, Taro, Çağatay, Tolga, Fujiwara, Ayano, Matsumura, Hiroyoshi, Niesman, Ashley B., Brautigam, Chad A., Chook, Yuh Min, and Yoshizawa, Takuya. Fri .
"Mechanism of karyopherin-β2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X)". United States. https://doi.org/10.1038/s41598-021-83196-y. https://www.osti.gov/servlets/purl/1772764.
@article{osti_1772764,
title = {Mechanism of karyopherin-β2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X)},
author = {Gonzalez, Abner and Mannen, Taro and Çağatay, Tolga and Fujiwara, Ayano and Matsumura, Hiroyoshi and Niesman, Ashley B. and Brautigam, Chad A. and Chook, Yuh Min and Yoshizawa, Takuya},
abstractNote = {Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid–liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-β2 (Kapβ2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapβ2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapβ2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapβ2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapβ2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapβ2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.},
doi = {10.1038/s41598-021-83196-y},
journal = {Scientific Reports},
number = 1,
volume = 11,
place = {United States},
year = {Fri Feb 12 00:00:00 EST 2021},
month = {Fri Feb 12 00:00:00 EST 2021}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins
journal, July 2018
- Wang, Jie; Choi, Jeong-Mo; Holehouse, Alex S.
- Cell, Vol. 174, Issue 3
ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules
journal, March 2013
- Vance, Caroline; Scotter, Emma L.; Nishimura, Agnes L.
- Human Molecular Genetics, Vol. 22, Issue 13
The Sam68 nuclear body is composed of two RNase-sensitive substructures joined by the adaptor HNRNPL
journal, July 2016
- Mannen, Taro; Yamashita, Seisuke; Tomita, Kozo
- Journal of Cell Biology, Vol. 214, Issue 1
Importazole, a Small Molecule Inhibitor of the Transport Receptor Importin-β
journal, April 2011
- Soderholm, Jonathan F.; Bird, Stephen L.; Kalab, Petr
- ACS Chemical Biology, Vol. 6, Issue 7
RGG/RG Motif Regions in RNA Binding and Phase Separation
journal, November 2018
- Chong, P. Andrew; Vernon, Robert M.; Forman-Kay, Julie D.
- Journal of Molecular Biology, Vol. 430, Issue 23
Monomethylated and unmethylated FUS exhibit increased binding to Transportin and distinguish FTLD-FUS from ALS-FUS
journal, February 2016
- Suárez-Calvet, Marc; Neumann, Manuela; Arzberger, Thomas
- Acta Neuropathologica, Vol. 131, Issue 4
Structural Basis for Cell-Cycle-Dependent Nuclear Import Mediated by the Karyopherin Kap121p
journal, June 2013
- Kobayashi, Junya; Matsuura, Yoshiyuki
- Journal of Molecular Biology, Vol. 425, Issue 11
Self-assembly of FUS through its low-complexity domain contributes to neurodegeneration
journal, February 2018
- Matsumoto, Taisei; Matsukawa, Koji; Watanabe, Naruaki
- Human Molecular Genetics, Vol. 27, Issue 8
ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import
journal, July 2010
- Dormann, Dorothee; Rodde, Ramona; Edbauer, Dieter
- The EMBO Journal, Vol. 29, Issue 16
Novel FUS/TLS Mutations and Pathology in Familial and Sporadic Amyotrophic Lateral Sclerosis
journal, April 2010
- Hewitt, Christopher; Kirby, Janine; Highley, J. Robin
- Archives of Neurology, Vol. 67, Issue 4
Clinical genetics of amyotrophic lateral sclerosis: what do we really know?
journal, October 2011
- Andersen, Peter M.; Al-Chalabi, Ammar
- Nature Reviews Neurology, Vol. 7, Issue 11
P525L FUS mutation is consistently associated with a severe form of juvenile Amyotrophic Lateral Sclerosis
journal, January 2012
- Conte, Amelia; Lattante, Serena; Zollino, Marcella
- Neuromuscular Disorders, Vol. 22, Issue 1
A juvenile sporadic amyotrophic lateral sclerosis case with P525L mutation in the FUS gene: A rare co-occurrence of autism spectrum disorder and tremor
journal, March 2019
- Eura, Nobuyuki; Sugie, Kazuma; Suzuki, Naoki
- Journal of the Neurological Sciences, Vol. 398
Arginine methylation by PRMT1 regulates nuclear-cytoplasmic localization and toxicity of FUS/TLS harbouring ALS-linked mutations
journal, September 2011
- Tradewell, Miranda L.; Yu, Zhenbao; Tibshirani, Michael
- Human Molecular Genetics, Vol. 21, Issue 1
FUS Recognizes G Quadruplex Structures Within Neuronal mRNAs
journal, February 2020
- Imperatore, Joshua A.; McAninch, Damian S.; Valdez-Sinon, Arielle N.
- Frontiers in Molecular Biosciences, Vol. 7
High-Precision Isothermal Titration Calorimetry with Automated Peak-Shape Analysis
journal, June 2012
- Keller, Sandro; Vargas, Carolyn; Zhao, Huaying
- Analytical Chemistry, Vol. 84, Issue 11
Structure of importin-β bound to the IBB domain of importin-α
journal, May 1999
- Cingolani, Gino; Petosa, Carlo; Weis, Karsten
- Nature, Vol. 399, Issue 6733
Juvenile ALS with basophilic inclusions is a FUS proteinopathy with FUS mutations
journal, July 2010
- Baumer, D.; Hilton, D.; Paine, S. M. L.
- Neurology, Vol. 75, Issue 7
The Effect of PRMT1-Mediated Arginine Methylation on the Subcellular Localization, Stress Granules, and Detergent-Insoluble Aggregates of FUS/TLS
journal, November 2012
- Yamaguchi, Atsushi; Kitajo, Keiko
- PLoS ONE, Vol. 7, Issue 11
XDS
journal, January 2010
- Kabsch, Wolfgang
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2
RNA-binding proteins with prion-like domains in health and disease
journal, April 2017
- Harrison, Alice Ford; Shorter, James
- Biochemical Journal, Vol. 474, Issue 8
Mutations in the FUS/TLS Gene on Chromosome 16 Cause Familial Amyotrophic Lateral Sclerosis
journal, February 2009
- Kwiatkowski, T. J.; Bosco, D. A.; LeClerc, A. L.
- Science, Vol. 323, Issue 5918
In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus
journal, January 2018
- Azevedo, Raimunda S. S.; de Sousa, Jorge R.; Araujo, Marialva T. F.
- Scientific Reports, Vol. 8, Issue 1
Treatment with a Global Methyltransferase Inhibitor Induces the Intranuclear Aggregation of ALS-Linked FUS Mutant In Vitro
journal, November 2015
- Fujii, Sakiko; Takanashi, Keisuke; Kitajo, Keiko
- Neurochemical Research, Vol. 41, Issue 4
Arginine methylation next to the PY-NLS modulates Transportin binding and nuclear import of FUS: Arginine methylation of FUS impairs TRN binding
journal, September 2012
- Dormann, Dorothee; Madl, Tobias; Valori, Chiara F.
- The EMBO Journal, Vol. 31, Issue 22
The regulation, functions and clinical relevance of arginine methylation
journal, July 2019
- Guccione, Ernesto; Richard, Stéphane
- Nature Reviews Molecular Cell Biology, Vol. 20, Issue 10
Design of Peptide Inhibitors for the Importin α/β Nuclear Import Pathway by Activity-Based Profiling
journal, September 2008
- Kosugi, Shunichi; Hasebe, Masako; Entani, Tetsuyuki
- Chemistry & Biology, Vol. 15, Issue 9
Defining the RGG/RG Motif
journal, June 2013
- Thandapani, Palaniraja; O’Connor, Timothy R.; Bailey, Timothy L.
- Molecular Cell, Vol. 50, Issue 5
Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: Application to adaptor protein complexes in cell signaling
journal, January 2007
- Houtman, Jon C. D.; Brown, Patrick H.; Bowden, Brent
- Protein Science, Vol. 16, Issue 1
Intrinsically disordered RGG/RG domains mediate degenerate specificity in RNA binding
journal, May 2017
- Ozdilek, Bagdeser A.; Thompson, Valery F.; Ahmed, Nasiha S.
- Nucleic Acids Research, Vol. 45, Issue 13
FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions
journal, April 2018
- Qamar, Seema; Wang, GuoZhen; Randle, Suzanne J.
- Cell, Vol. 173, Issue 3
Correlation of CRM1-NES affinity with nuclear export activity
journal, August 2018
- Fu, Szu-Chin; Fung, Ho Yee Joyce; Cağatay, Tolga
- Molecular Biology of the Cell, Vol. 29, Issue 17
Frameshift and novel mutations in FUS in familial amyotrophic lateral sclerosis and ALS/dementia
journal, July 2010
- Yan, J.; Deng, H. - X.; Siddique, N.
- Neurology, Vol. 75, Issue 9
Structure of the nuclear transport complex karyopherin-β2–Ran˙GppNHp
journal, May 1999
- Chook, Yuh Min; Blobel, Günter
- Nature, Vol. 399, Issue 6733
The Solution Structure of FUS Bound to RNA Reveals a Bipartite Mode of RNA Recognition with Both Sequence and Shape Specificity
journal, February 2019
- Loughlin, Fionna E.; Lukavsky, Peter J.; Kazeeva, Tamara
- Molecular Cell, Vol. 73, Issue 3
MolProbity : all-atom structure validation for macromolecular crystallography
journal, December 2009
- Chen, Vincent B.; Arendall, W. Bryan; Headd, Jeffrey J.
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1
Intracellular localization and splicing regulation of FUS/TLS are variably affected by amyotrophic lateral sclerosis-linked mutations
journal, November 2010
- Kino, Yoshihiro; Washizu, Chika; Aquilanti, Elisa
- Nucleic Acids Research, Vol. 39, Issue 7
Features and development of Coot
journal, March 2010
- Emsley, P.; Lohkamp, B.; Scott, W. G.
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
Super-Resolution Microscopy Reveals Presynaptic Localization of the ALS/FTD Related Protein FUS in Hippocampal Neurons
journal, January 2016
- Schoen, Michael; Reichel, Jochen M.; Demestre, Maria
- Frontiers in Cellular Neuroscience, Vol. 9
Rules for Nuclear Localization Sequence Recognition by Karyopherinβ2
journal, August 2006
- Lee, Brittany J.; Cansizoglu, Ahmet E.; Süel, Katherine E.
- Cell, Vol. 126, Issue 3
PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010
- Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
Calculations and Publication-Quality Illustrations for Analytical Ultracentrifugation Data
book, January 2015
- Brautigam, Chad A.
- Methods in Enzymology
Direct visualization of the conformational change of FUS/TLS upon binding to promoter-associated non-coding RNA
journal, January 2020
- Hamad, Nesreen; Watanabe, Hiroki; Uchihashi, Takayuki
- Chemical Communications, Vol. 56, Issue 64
Specific Binding of Modified RGG Domain in TLS/FUS to G-Quadruplex RNA: Tyrosines in RGG Domain Recognize 2′-OH of the Riboses of Loops in G-Quadruplex
journal, November 2013
- Takahama, Kentaro; Oyoshi, Takanori
- Journal of the American Chemical Society, Vol. 135, Issue 48
Nonclassical nuclear localization signals mediate nuclear import of CIRBP
journal, March 2020
- Bourgeois, Benjamin; Hutten, Saskia; Gottschalk, Benjamin
- Proceedings of the National Academy of Sciences, Vol. 117, Issue 15
Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules
journal, August 2010
- Bosco, Daryl A.; Lemay, Nathan; Ko, Hae Kyung
- Human Molecular Genetics, Vol. 19, Issue 21
Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites
journal, April 2018
- Yoshizawa, Takuya; Ali, Rustam; Jiou, Jenny
- Cell, Vol. 173, Issue 3
Importin-9 wraps around the H2A-H2B core to act as nuclear importer and histone chaperone
journal, March 2019
- Padavannil, Abhilash; Sarkar, Prithwijit; Kim, Seung Joong
- eLife, Vol. 8
Arginine Methylation: The Coming of Age
journal, January 2017
- Blanc, Roméo S.; Richard, Stéphane
- Molecular Cell, Vol. 65, Issue 1
Structural and energetic basis of ALS-causing mutations in the atypical proline–tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)
journal, July 2012
- Zhang, Zi Chao; Chook, Yuh Min
- Proceedings of the National Academy of Sciences, Vol. 109, Issue 30
The RNA Binding Protein TLS Is Translocated to Dendritic Spines by mGluR5 Activation and Regulates Spine Morphology
journal, March 2005
- Fujii, Ritsuko; Okabe, Shigeo; Urushido, Tomoe
- Current Biology, Vol. 15, Issue 6
Structure-based design of a pathway-specific nuclear import inhibitor
journal, April 2007
- Cansizoglu, Ahmet E.; Lee, Brittany J.; Zhang, Zi Chao
- Nature Structural & Molecular Biology, Vol. 14, Issue 5
Detection of Arginine Dimethylated Peptides by Parallel Precursor Ion Scanning Mass Spectrometry in Positive Ion Mode
journal, July 2003
- Rappsilber, Juri; Friesen, Westley J.; Paushkin, Sergey
- Analytical Chemistry, Vol. 75, Issue 13
Novel missense and truncating mutations in FUS/TLS in familial ALS
journal, July 2010
- Waibel, S.; Neumann, M.; Rabe, M.
- Neurology, Vol. 75, Issue 9
Phaser crystallographic software
journal, July 2007
- McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
- Journal of Applied Crystallography, Vol. 40, Issue 4
Functions of FUS/TLS From DNA Repair to Stress Response: Implications for ALS
journal, June 2014
- Sama, Reddy Ranjith Kumar; Ward, Catherine L.; Bosco, Daryl A.
- ASN Neuro, Vol. 6, Issue 4