@article{53b438bf211e4ff68a23b15490a2931c,
title = "Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites",
abstract = "Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-β2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-β2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-β2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-β2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-β2 may act analogously to control condensates in diverse cellular contexts. Distributed, energetically weak interactions between a karyopherin and the FUS nuclear localization signal disrupt FUS phase separation.",
keywords = "FUS, PY-NLS, RNA granule, amyotrophic lateral sclerosis, biomolecular condensate, intrinsically disordered protein, karyopherin-β2, liquid-liquid phase separation, low-complexity sequences, transportin-1",
author = "Takuya Yoshizawa and Rustam Ali and Jenny Jiou and Fung, {Ho Yee Joyce} and Burke, {Kathleen A.} and Kim, {Seung Joong} and Yuan Lin and Peeples, {William B.} and Daniel Saltzberg and Michael Soniat and Baumhardt, {Jordan M.} and Rudolf Oldenbourg and Andrej Sali and Fawzi, {Nicolas L.} and Rosen, {Michael K.} and Chook, {Yuh Min}",
note = "Funding Information: We thank D. Dormann and J. Shorter for critical reading of the manuscript. We thank A. Patel and A. A. Hyman for their gift of FUS-GFP, T. Cagatay and A. Gonzalez for help subcloning, J. Hu and D. Corey for their help with temperature-dependent turbidity analysis, N. Conrad and K. Lynch for advice on FUS-RNA analysis, T. Matsui and T.M. Weiss at SSRL, SLAC National Accelerator Laboratory, for assistance with collecting SAXS data, and B. Raveh for discussions on SAXS analysis. We thank D. Tomchick for advice on X-ray structure determination, and the Structural Biology Laboratory and Macromolecular Biophysics Resource at UTSW for their assistance with crystallographic and biophysical data collection. Crystallographic results are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the U.S. Department of Energy (DOE), Office of Biological and Environmental Research (BER) under contract DE-AC02-06CH11357. NMR data were obtained in part at the Brown University Structural Biology Core Facility supported by the Division of Biology and Medicine, Brown University. NMR spectroscopy at UTSW is supported by NIH instrumentation grants 1S10RR26461-1 and 1S10OD018027-01. SAXS experiments were performed at the SSRL, SLAC National Accelerator Laboratory operated for DOE by Stanford University. The SSRL SMBP is supported by the DOE BER, by the NIH, NCRR, Biomedical Technology Program (P41RR001209), and by NIGMS, NIH (P41GM103393). This work was funded by NIGMS of NIH under R01GM069909 (Y.M.C.), U01GM98256-01 (Y.M.C. and A.S.), R01GM56322 (M.K.R.), R01GM118530 (N.L.F.), R01GM083960, P41GM109824, and R01GM112108 (A.S), R01GM114274 (R.O.), T32GM008203 (J.J.), T32GM008297 (J.M.B.), the Howard Hughes Medical Institute HCIA grant (M.K.R.), the Welch Foundation grants I-1532 (Y.M.C.) and I-1544 (M.K.R.), Leukemia and Lymphoma Society Scholar Award (Y.M.C.), and the University of Texas Southwestern Endowed Scholars Program (Y.M.C.). Work in M.K.R.{\textquoteright}s lab was supported by the Howard Hughes Medical Institute. R.A. was an American Heart Association Postdoctoral Fellow and H.Y.J.F was a Howard Hughes Medical Institute International Student Research fellow. Funding Information: We thank D. Dormann and J. Shorter for critical reading of the manuscript. We thank A. Patel and A. A. Hyman for their gift of FUS-GFP, T. Cagatay and A. Gonzalez for help subcloning, J. Hu and D. Corey for their help with temperature-dependent turbidity analysis, N. Conrad and K. Lynch for advice on FUS-RNA analysis, T. Matsui and T.M. Weiss at SSRL, SLAC National Accelerator Laboratory, for assistance with collecting SAXS data, and B. Raveh for discussions on SAXS analysis. We thank D. Tomchick for advice on X-ray structure determination, and the Structural Biology Laboratory and Macromolecular Biophysics Resource at UTSW for their assistance with crystallographic and biophysical data collection. Crystallographic results are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the U.S. Department of Energy (DOE), Office of Biological and Environmental Research (BER) under contract DE-AC02-06CH11357 . NMR data were obtained in part at the Brown University Structural Biology Core Facility supported by the Division of Biology and Medicine, Brown University . NMR spectroscopy at UTSW is supported by NIH instrumentation grants 1S10RR26461-1 and 1S10OD018027-01 . SAXS experiments were performed at the SSRL, SLAC National Accelerator Laboratory operated for DOE by Stanford University. The SSRL SMBP is supported by the DOE BER , by the NIH, NCRR , Biomedical Technology Program ( P41RR001209 ), and by NIGMS, NIH ( P41GM103393 ). This work was funded by NIGMS of NIH under R01GM069909 (Y.M.C.), U01GM98256-01 (Y.M.C. and A.S.), R01GM56322 (M.K.R.), R01GM118530 (N.L.F.), R01GM083960 , P41GM109824 , and R01GM112108 (A.S), R01GM114274 (R.O.), T32GM008203 (J.J.), T32GM008297 (J.M.B.), the Howard Hughes Medical Institute HCIA grant (M.K.R.), the Welch Foundation grants I-1532 (Y.M.C.) and I-1544 (M.K.R.), Leukemia and Lymphoma Society Scholar Award (Y.M.C.), and the University of Texas Southwestern Endowed Scholars Program (Y.M.C.). Work in M.K.R.{\textquoteright}s lab was supported by the Howard Hughes Medical Institute . R.A. was an American Heart Association Postdoctoral Fellow and H.Y.J.F was a Howard Hughes Medical Institute International Student Research fellow. Publisher Copyright: {\textcopyright} 2018 Elsevier Inc.",
year = "2018",
month = apr,
day = "19",
doi = "10.1016/j.cell.2018.03.003",
language = "English (US)",
volume = "173",
pages = "693--705.e22",
journal = "Cell",
issn = "0092-8674",
publisher = "Cell Press",
number = "3",
}