ALS mutations of FUS suppress protein translation and disrupt the regulation of nonsense-mediated decay Marisa Kamelgarna, Jing Chenb, Lisha Kuangb, Huan Jina, Edward J. Kasarskisa,c, and Haining Zhua,b,d,1 aDepartment of Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536; bDepartment of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY 40536; cDepartment of Neurology, College of Medicine, University of Kentucky, Lexington, KY 40536; and dLexington VA Medical Center, Research and Development, Lexington, KY 40502 Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved October 22, 2018 (received for review June 16, 2018) Amyotrophic lateral sclerosis (ALS) is an incurable neurodegener- cytoplasmic granules is likely to provide critical insights into the ative disease characterized by preferential motor neuron death. toxic mechanism of mutant FUS. We developed a protocol to Approximately 15% of ALS cases are familial, and mutations in the capture the dynamic mutant FUS-positive granules (3, 4) by fused in sarcoma (FUS) gene contribute to a subset of familial ALS membrane filtration and identified protein components by pro- cases. FUS is a multifunctional protein participating in many RNA teomic approaches. The bioinformatics analysis of proteins identi- metabolism pathways. ALS-linked mutations cause a liquid–liquid fied in wild-type (WT) and mutant FUS granules revealed multiple phase separation of FUS protein in vitro, inducing the formation of RNA metabolism pathways, among which protein translation and cytoplasmic granules and inclusions. However, it remains elusive mRNA surveillance appeared to be novel. what other proteins are sequestered into the inclusions and how We thus hypothesize that mutant FUS plays a role in protein such a process leads to neuronal dysfunction and degeneration. In translation. Two previous studies reported that ALS-linked FUS this study, we developed a protocol to isolate the dynamic mutant mutations were recruited to ribonucleoprotein granules; thus, FUS FUS-positive cytoplasmic granules. Proteomic identification of the was speculated to be involved in protein translation (3, 20). How- protein composition and subsequent pathway analysis led us to ever, protein translation was not measured in either study. Using hypothesize that mutant FUS can interfere with protein transla- three independent assays, we found that mutant FUS indeed im- tion. We demonstrated that the ALS mutations in FUS indeed sup- paired protein translation and that the cytoplasmic inclusions of pressed protein translation in N2a cells expressing mutant FUS and fibroblast cells derived from FUS ALS cases. In addition, the mutant FUS were positive for stalled ribosomal complexes. nonsense-mediated decay (NMD) pathway, which is closely related Mutations in FUS have been demonstrated to cause aberrant to protein translation, was altered by mutant FUS. Specifically, splicing (21); however, the molecular mechanism by which cells NMD-promoting factors UPF1 and UPF3b increased, whereas a handle defective mRNA has not been explored in ALS. negative NMD regulator, UPF3a, decreased, leading to the disrup- Nonsense-mediated decay (NMD) is a major mRNA surveil- ∼ – tion of NMD autoregulation and the hyperactivation of NMD. Al- lance system that is known to degrade defective mRNA and 3 terations in NMD factors and elevated activity were also observed 20% of all mRNAs (22). NMD and protein translation are in- in the fibroblast cells of FUS ALS cases. We conclude that mutant terrelated, as NMD utilizes the translocating ribosome as a FUS suppresses protein biosynthesis and disrupts NMD regulation, proofreading mechanism for sensing defective mRNAs (23–25). both of which likely contribute to motor neuron death. We demonstrate that the phosphorylation level of a critical NMD regulator UPF1 (24), the NMD complex assembly, and the amyotrophic lateral sclerosis | fused in sarcoma | protein translation | nonsense-mediated decay | RNA-protein granules Significance myotrophic lateral sclerosis (ALS) is a neurodegenerative Mutations in fused in sarcoma (FUS) contribute to a subset of Adisease characterized by motor neuron death and progressive familial amyotrophic lateral sclerosis (ALS). ALS-linked mutations muscle wasting and paralysis. Approximately 15% of ALS cases are cause a liquid–liquid phase separation of FUS protein, induce caused by inheritable genetic mutations, mostly in an autosomal cytoplasmic inclusions in cells, and interfere with RNA metabo- dominant fashion. Mutations in the fused in sarcoma (FUS)gene lism pathways. Our proteomic analysis shows that proteins se- were discovered to contribute to a subset of familial ALS (1, 2). questered into inclusions are enriched in translation and RNA FUS is a DNA- and RNA-binding protein that is primarily lo- quality surveillance pathways. This study demonstrates that ALS calized to the nucleus, where it forms dynamic ribonucleoprotein mutations in FUS indeed suppress protein translation and affect granules. In contrast, ALS-related mutant FUS accumulates in the the mRNA nonsense-mediated decay (NMD) pathway. NMD- cytoplasm and forms stable ribonucleoprotein granules, which can promoting factors UPF1 and UPF3b increased, whereas the nega- lead to inclusion bodies and potentially contribute to neurotoxicity tive regulator UPF3a decreased in the cells of patients with (3–5). FUS mutations have also been shown to impact many RNA ALS. The disruption in NMD regulation and suppression of pro- metabolic processes, including transcription (6–8), splicing (9–11), tein biosynthesis likely contribute to neurodegeneration in ALS. mRNA transport (12), and stabilization (13), ultimately contributing Author contributions: M.K. and H.Z. designed research; M.K., J.C., L.K., and H.J. performed to neuronal dysfunction. Recent studies demonstrated that muta- research; E.J.K. contributed new reagents/analytic tools; M.K., J.C., L.K., H.J., E.J.K., and tions in FUS cause the liquid–liquid phase separation (LLPS) of H.Z. analyzed data; and M.K. and H.Z. wrote the paper. FUS protein and the formation of self-assembled hydrogels (14) or The authors declare no conflict of interest. liquid droplets in vitro (15, 16). It is noted that LLPS has also been This article is a PNAS Direct Submission. reported for other RNA metabolic proteins involved in ALS, in- This open access article is distributed under Creative Commons Attribution-NonCommercial- cluding TDP-43 (17), C9ORF72 dipeptide repeat (18), hnRNPA1 NoDerivatives License 4.0 (CC BY-NC-ND). (17), and TIA1 (19). Thus, other cellular proteins are likely to be See Commentary on page 12842. included in granules during LLPS in living cells, but the identities of 1To whom correspondence should be addressed. Email: [email protected]. such proteins remain to be determined. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. This study started with testing the hypothesis that the iden- 1073/pnas.1810413115/-/DCSupplemental. tification of proteins associated with mutant FUS-dependent Published online November 19, 2018. E11904–E11913 | PNAS | vol. 115 | no. 51 www.pnas.org/cgi/doi/10.1073/pnas.1810413115 Downloaded by guest on September 30, 2021 UPF1-mRNA binding all increased in the presence of mutant therefore focused on testing the function of FUS in protein syn- FUS, supporting that NMD is activated by mutant FUS. Addi- thesis and related mRNA surveillance pathways. SEE COMMENTARY tionally, two potent NMD-activating regulators [UPF1 and We next examined the subcellular localization of critical proteins UPF3b (24, 26, 27)] were up-regulated, while the negative NMD involved in protein translation (eIF3, eIF4A1, eIF4G, and rpS6) regulator [UPF3a (28)] was down-regulated in skin fibroblast and mRNA surveillance (eIF4A3) that were identified in the MS cells derived from a cohort of patients with ALS with FUS mu- results. In primary cortical neurons transfected with EGFP-tagged tations compared with cells from control subjects, indicating FUS, eIF4A3 (Fig. 1E), eIF3 (Fig. 1F), eIF4A1, eIF4G, and rpS6 disruption of the autoregulation of NMD. The hyperactivation of (SI Appendix,Fig.S3A–C, respectively) were all colocalized with NMD was demonstrated using an NMD reporter assay in N2a cytoplasmic inclusions of mutant FUS. As a positive control, mu- cells and measuring endogenous mRNAs in the fibroblast cells of tant FUS inclusions in primary neurons were positive for the stress FUS ALS cases. Overall, the findings from this study thus pro- granule marker G3BP1 (SI Appendix,Fig.S3D)aspreviously vide an in-depth understanding of how RNA metabolism and reported (4). The immunoprecipitation (IP) results also demon- protein translation are impacted by mutations in FUS and pro- strated that eIF3, eIF4G, and eIF4A3 interacted more with the duce insights into the disease-causing mechanism of the mutant mutant than WT FUS (Fig. 1G), validating the proteomic and FUS subtype of ALS. colocalization results. Results Protein Translation Is Impaired in the Presence of Mutant FUS. Based Proteins Related to Translation and mRNA Surveillance Are Enriched on the above proteomic identifications, GO enrichment analysis, in Mutant FUS Inclusions. A more complete understanding of the and colocalization of translation machinery to the mutant FUS protein composition that makes up the inclusions characteristic inclusions, we set out to test whether protein