Abstract
Repeat expansion in the C9orf72 gene is the most common cause of the neurodegenerative disorder amyotrophic lateral sclerosis (C9-ALS) and is linked to the unconventional translation of five dipeptide-repeat polypeptides (DPRs). The two enriched in arginine, poly(GR) and poly(PR), infiltrate liquid-like nucleoli, co-localize with the nucleolar protein nucleophosmin (NPM1), and alter the phase separation behavior of NPM1 in vitro. Here, we show that poly(PR) DPRs bind tightly to a long acidic tract within the intrinsically disordered region of NPM1, altering its phase separation with nucleolar partners to the extreme of forming large, soluble complexes that cause droplet dissolution in vitro. In cells, poly(PR) DPRs disperse NPM1 from nucleoli and entrap rRNA in static condensates in a DPR-length-dependent manner. We propose that R-rich DPR toxicity involves disrupting the role of phase separation by NPM1 in organizing ribosomal proteins and RNAs within the nucleolus. ALS-associated dipeptide repeats (R-rich DPRs) alter NPM1 phase separation, leading to NPM1 sequestration and driving droplet dissolution in vitro and NPM1 delocalization from nucleoli. Further, poly(PR) DPRs entrap rRNA within static puncta in vitro and in nucleoli. We propose that disruption of nucleolar liquid-phase homeostasis contributes to arginine-rich DPR toxicity.
Original language | English |
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Pages (from-to) | 713-728.e6 |
Journal | Molecular Cell |
Volume | 74 |
Issue number | 4 |
DOIs | |
State | Published - May 16 2019 |
Funding
We thank Mr. Cheon-Gil Park, Mrs. Nazia Ahmed, and Dr. Maxime Cuypers for assistance with cloning, protein purification, and SANS data collection, respectively. We also thank the Hartwell Center at SJCRH and Dr. Patrick Rodrigues, who synthesized peptides of ≥30 PR and GR repeats. We thank Maxim Mayzel, Helene Kovacs, and Rainer Kummerle for assistance with 1.1 GHz NMR measurements at Bruker Biospin AG in Faellanden, Switzerland. Fluorescence microscopy images were acquired at the Cell & Tissue Imaging Center at SJCRH, supported by SJCRH and the NIH ( NCI P30 CA021765 ), with additional assistance from Dr. Aaron Pitre. SAS measurements were performed at ORNL’s Spallation Neutron Source, sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, US DOE . M.R.W. acknowledges support from the NIH ( NIGMS F32GM131668-01 ). D.E.C. acknowledges support from the NIH ( NCI R25CA23944 ). R.W.K. acknowledges support from the NIH ( NIGMS 1R01GM115634 ). J.P.T. acknowledges support from the NIH ( NINDS R35NS097974 ) and the Howard Hughes Medical Institute (HHMI). Both R.W.K. and J.P.T. acknowledge support from St. Jude Research Collaborative on Membrane-less Organelles and ALSAC . We thank Mr. Cheon-Gil Park, Mrs. Nazia Ahmed, and Dr. Maxime Cuypers for assistance with cloning, protein purification, and SANS data collection, respectively. We also thank the Hartwell Center at SJCRH and Dr. Patrick Rodrigues, who synthesized peptides of ≥30 PR and GR repeats. We thank Maxim Mayzel, Helene Kovacs, and Rainer Kummerle for assistance with 1.1 GHz NMR measurements at Bruker Biospin AG in Faellanden, Switzerland. Fluorescence microscopy images were acquired at the Cell & Tissue Imaging Center at SJCRH, supported by SJCRH and the NIH (NCI P30 CA021765), with additional assistance from Dr. Aaron Pitre. SAS measurements were performed at ORNL's Spallation Neutron Source, sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, US DOE. M.R.W. acknowledges support from the NIH (NIGMS F32GM131668-01). D.E.C. acknowledges support from the NIH (NCI R25CA23944). R.W.K. acknowledges support from the NIH (NIGMS 1R01GM115634). J.P.T. acknowledges support from the NIH (NINDS R35NS097974) and the Howard Hughes Medical Institute (HHMI). Both R.W.K. and J.P.T. acknowledge support from St. Jude Research Collaborative on Membrane-less Organelles and ALSAC. R.W.K. D.M.M. and M.R.W. conceived the project, designed experiments, and interpreted all data. A.H.P. analyzed and interpreted NMR data. D.E.C. performed turbidity and microscopy analyses comparing NPM1 and NPM1 A-tract mutant constructs with M.R.W. Data collection and reduction from X-ray and neutron scattering experiments were performed by C.B.S. and analyzed by C.B.S. and M.R.W. A.N. performed, analyzed, and interpreted AUC experiments. M.T. purified E. coli rRNA. J.P.T. and P.Z. designed in-cell experiments, with input from R.W.K. and M.R.W. that were performed by P.Z. and M.R.W. Analysis of cell assays was performed by P.Z. and M.R.W. and data were interpreted by J.P.T. and P.Z. with input from R.W.K, D.M.M. and M.R.W. Data from all other experiments were collected and analyzed by M.R.W. M.R.W wrote the manuscript with R.W.K. and D.M.M. All authors edited the manuscript. The authors declare no competing interests.
Keywords
- ALS
- C9orf72
- DPR
- RNA
- dipeptide repeat
- intrinsically disordered region
- liquid-liquid phase separation
- nucleolus
- nucleophosmin
- ribosome