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ALS Mutations of FUS Suppress Protein Translation and Disrupt the Regulation of Nonsense-Mediated Decay

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ALS Mutations of FUS Suppress Protein Translation and Disrupt the Regulation of Nonsense-Mediated Decay 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- This study started with testing the hypothesis that the identi- ative disease characterized by preferential motor neuron death. fication of proteins associated with mutant FUS-dependent cy- Approximately 15% of ALS cases are familial, and mutations in the toplasmic granules is likely to provide critical insights into the fused in sarcoma (FUS) gene contribute to a subset of familial ALS toxic mechanism of mutant FUS. We developed a protocol to cases. FUS is a multifunctional protein participating in many RNA capture the dynamic mutant FUS-positive granules (3, 4) by metabolism pathways. ALS-linked mutations cause a liquid–liquid membrane filtration and identified protein components by pro- phase separation of FUS protein in vitro, inducing the formation of teomic approaches. The bioinformatics analysis of proteins cytoplasmic granules and inclusions. However, it remains elusive identified in wild-type (WT) and mutant FUS granules revealed what other proteins are sequestered into the inclusions and how multiple RNA metabolism pathways, among which protein such a process leads to neuronal dysfunction and degeneration. In translation and mRNA surveillance appeared to be novel. this study, we developed a protocol to isolate the dynamic mutant We thus hypothesize that mutant FUS plays a role in protein FUS-positive cytoplasmic granules. Proteomic identification of the translation. Two previous studies reported that ALS-linked FUS protein composition and subsequent pathway analysis led us to mutations were recruited to ribonucleoprotein granules; thus, FUS hypothesize that mutant FUS can interfere with protein transla- was speculated to be involved in protein translation (3, 20). How- tion. We demonstrated that the ALS mutations in FUS indeed sup- ever, protein translation was not measured in either study. Using pressed protein translation in N2a cells expressing mutant FUS and three independent assays, we found that mutant FUS indeed im- BIOCHEMISTRY fibroblast cells derived from FUS ALS cases. In addition, the paired protein translation and that the cytoplasmic inclusions of nonsense-mediated decay (NMD) pathway, which is closely related mutant FUS were positive for stalled ribosomal complexes. to protein translation, was altered by mutant FUS. Specifically, Mutations in FUS have been demonstrated to cause aberrant NMD-promoting factors UPF1 and UPF3b increased, whereas a splicing (21); however, the molecular mechanism by which cells negative NMD regulator, UPF3a, decreased, leading to the disrup- handle defective mRNA has not been explored in ALS. tion of NMD autoregulation and the hyperactivation of NMD. Al- Nonsense-mediated decay (NMD) is a major mRNA surveil- terations in NMD factors and elevated activity were also observed lance system that is known to degrade defective mRNA and ∼3– in the fibroblast cells of FUS ALS cases. We conclude that mutant 20% of all mRNAs (22). NMD and protein translation are in- FUS suppresses protein biosynthesis and disrupts NMD regulation, terrelated, as NMD utilizes the translocating ribosome as a both of which likely contribute to motor neuron death. proofreading mechanism for sensing defective mRNAs (23–25). We demonstrate that the phosphorylation level of a critical amyotrophic lateral sclerosis | fused in sarcoma | protein translation | NMD regulator UPF1 (24), the NMD complex assembly, and the nonsense-mediated decay | RNA-protein granules Significance myotrophic lateral sclerosis (ALS) is a neurodegenerative Adisease characterized by motor neuron death and pro- Mutations in fused in sarcoma (FUS) contribute to a subset of gressive muscle wasting and paralysis. Approximately 15% of familial amyotrophic lateral sclerosis (ALS). ALS-linked mutations ALS cases are caused by inheritable genetic mutations, mostly in cause a liquid–liquid phase separation of FUS protein, induce an autosomal dominant fashion. Mutations in the fused in sar- cytoplasmic inclusions in cells, and interfere with RNA metabo- coma (FUS) gene were discovered to contribute to a subset of lism pathways. Our proteomic analysis shows that proteins se- familial ALS (1, 2). questered into inclusions are enriched in translation and RNA FUS is a DNA- and RNA-binding protein that is primarily quality surveillance pathways. This study demonstrates that ALS localized to the nucleus, where it forms dynamic ribonucleo- mutations in FUS indeed suppress protein translation and affect protein granules. In contrast, ALS-related mutant FUS accu- the mRNA nonsense-mediated decay (NMD) pathway. NMD- mulates in the cytoplasm and forms stable ribonucleoprotein promoting factors UPF1 and UPF3b increased, whereas the nega- granules, which can lead to inclusion bodies and potentially tive regulator UPF3a decreased in the cells of patients with contribute to neurotoxicity (3–5). FUS mutations have also been ALS. The disruption in NMD regulation and suppression of pro- shown to impact many RNA metabolic processes, including tein biosynthesis likely contribute to neurodegeneration in ALS. transcription (6–8), splicing (9–11), mRNA transport (12), and Author contributions: M.K. and H.Z. designed research; M.K., J.C., L.K., and H.J. performed stabilization (13), ultimately contributing to neuronal dysfunc- research; E.J.K. contributed new reagents/analytic tools; M.K., J.C., L.K., H.J., E.J.K., and tion. Recent studies demonstrated that mutations in FUS cause H.Z. analyzed data; and M.K. and H.Z. wrote the paper. the liquid–liquid phase separation (LLPS) of FUS protein and The authors declare no conflict of interest. the formation of self-assembled hydrogels (14) or liquid droplets This article is a PNAS Direct Submission. in vitro (15, 16). It is noted that LLPS has also been reported for This open access article is distributed under Creative Commons Attribution-NonCommercial- other RNA metabolic proteins involved in ALS, including TDP- NoDerivatives License 4.0 (CC BY-NC-ND). 43 (17), C9ORF72 dipeptide repeat (18), hnRNPA1 (17), and 1To whom correspondence should be addressed. Email: [email protected]. TIA1 (19). Thus, other cellular proteins are likely to be included This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in granules during LLPS in living cells, but the identities of such 1073/pnas.1810413115/-/DCSupplemental. proteins remain to be determined. www.pnas.org/cgi/doi/10.1073/pnas.1810413115 PNAS Latest Articles | 1of10 Downloaded by guest on September 25, 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. 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,andrpS6 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 translation is im- of mutant FUS-dependent ALS would provide a better un-
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