A Deletion of the Nuclear Localization Signal Domain in the Fus Protein Induces Stable Post-stress Cytoplasmic Inclusions in SH-SY5Y Cells

Mutations in Fused-in-Sarcoma (FUS) gene involving the nuclear localization signal (NLS) domain lead to juvenile-onset Amyotrophic Lateral Sclerosis (ALS). The mutant protein mislocalizes to the cytoplasm, incorporating it into Stress Granules (SG). Whether SGs are the first step to the formation of stable FUS-containing aggregates is still unclear. In this work, we used acute and chronic stress paradigms to study the SG dynamics in a human SH-SY5Y neuroblastoma cell line carrying a deletion of the NLS domain of the FUS protein (homozygous: ΔNLS–/–; heterozygous: ΔNLS+/–). Wild-type (WT) cells served as controls. We evaluated the subcellular localization of the mutant protein through immunoblot and immunofluorescence, in basal conditions and after acute stress and chronic stress with sodium arsenite (NaAsO2). Cells were monitored for up to 24 h after rescue. FUS was expressed in both nucleus and cytoplasm in the ΔNLS+/– cells, whereas it was primarily cytoplasmic in the ΔNLS–/–. Acute NaAsO2 exposure induced SGs: at rescue,>90% of ΔNLS cells showed abundant FUS-containing if compared to less than 5% of the WT cells. The proportion of FUS-positive SGs remained 15–20% at 24 h in mutant cells. Cycloheximide did not abolish the long-lasting SGs in mutant cells. Chronic exposure to NaAsO2 did not induce significant SGs formation. A wealth of research has demonstrated that ALS-associated FUS mutations at the C-terminus facilitate the incorporation of the mutant protein into SGs. We have shown here that mutant FUS-containing SGs tend to fail to dissolve after stress, facilitating a liquid-to-solid phase transition. The FUS-containing inclusions seen in the dying motor neurons might therefore directly derive from SGs. This might represent an attractive target for future innovative therapies.

Long noncoding RNA SOX2OT promotes pancreatic cancer cell migration and invasion through destabilizing FUS protein via ubiquitination

Pancreatic cancer is a highly aggressive and lethal digestive system malignancy. Our previous studies revealed the correlation of high levels of lncRNA SOX2OT expression with patients’ poor survival outcomes, the promoting role of SOX2OT in proliferation and cycle progression of pancreatic cancer cells, and the in vivo binding of SOX2OT to RNA binding protein FUS, which destabilized the protein expression of FUS. However, the mechanism of SOX2OT binding and inhibiting FUS protein stability remains unclear. In this study, we performed RNA pull-down, cycloheximide-chase, and ubiquitination assays to determine the effect of SOX2OT on FUS ubiquitination, and explored the specific regulatory mechanism of SOX2OT–FUS axis in pancreatic cancer cell migration, invasion, in vivo tumor growth, and metastasis through RNA sequencing. We found that SOX2OT binds to FUS through its 5′ and 3′ regions, resulting in FUS ubiquitination and degradation. The SOX2OT–FUS regulatory axis promotes migration, invasion, tumor growth, and metastasis ability of pancreatic cancer cells. The in-depth elaboration of the SOX2OT–FUS regulatory axis in pancreatic cancer may clarify the mechanism of action of SOX2OT and provide new ideas for pancreatic cancer treatment.

Wild-type FUS corrects ALS-like disease induced by cytoplasmic mutant FUS through autoregulation

Mutations in FUS, an RNA-binding protein involved in multiple steps of RNA metabolism, are associated with the most severe forms of amyotrophic lateral sclerosis (ALS). Accumulation of cytoplasmic FUS is likely to be a major culprit in the toxicity of FUS mutations. Thus, preventing cytoplasmic mislocalization of the FUS protein may represent a valuable therapeutic strategy. FUS binds to its own pre-mRNA creating an autoregulatory loop efficiently buffering FUS excess through multiple proposed mechanisms including retention of introns 6 and/or 7. Here, we introduced a wild-type FUS gene allele, retaining all intronic sequences, in mice whose heterozygous or homozygous expression of a cytoplasmically retained FUS protein (Fus∆NLS) was previously shown to provoke ALS-like disease or postnatal lethality, respectively. Wild-type FUS completely rescued the early lethality caused by the two Fus∆NLS alleles, and improved the age-dependent motor deficits and reduced lifespan caused by heterozygous expression of mutant FUS∆NLS. Mechanistically, wild-type FUS decreased the load of cytoplasmic FUS, increased retention of introns 6 and 7 in the endogenous mouse Fus mRNA, and decreased expression of the mutant mRNA. Thus, the wild-type FUS allele activates the homeostatic autoregulatory loop, maintaining constant FUS levels and decreasing the mutant protein in the cytoplasm. These results provide proof of concept that an autoregulatory competent wild-type FUS expression could protect against this devastating, currently intractable, neurodegenerative disease.

Fused in Sarcoma (FUS) Expression May Predict Survival Outcomes of Patients With Advanced Squamous Cell Cervical Carcinoma

Because of its role in preserving DNA/RNA stability, there is an increasing number of studies addressing the relationship between Fused in Sarcoma (FUS) expression and cancer. However, the disparity in FUS/FUS oncogenic vs. tumor-suppressive roles may be attributed to the complex molecular pathways associated with FUS regulation in different cancer types, and its role in cervical carcinogenesis remains largely unexplored. Methods: We determined FUS protein expression in specimens of 61 patients with advanced cervical cancer. Long-term (> 10 years) clinical follow-up data for these patients were available, and we determined disease-free, cancer-related and overall survival as related to FUS expression. Results: There were no significant associations between FUS expression and patients’ age, tumor grade, and acute/late toxicity events related to treatment (either radiation alone or chemoradiation). However, multivariate Cox regression analysis for disease-free survival (recurrence), overall survival (death) and cancer-related survival showed that patients with high average FUS expression fared significantly better than their counterparts with low average FUS expression, both in terms of disease-free survival (HR = 0.31; 95%CI 0.12 to 0.77; p = 0.01) and cancer-related survival (HR = 0.41; 95%CI 0.17 to 0.98; p = 0.04). Conclusions: Our study shows that high FUS protein expression in advanced cervical cancer specimens is a potent harbinger of better prognosis, and can as such be used in clinical practice to help characterize patients and, possibly, plan treatment and follow-up strategies.

Retinal abnormalities in transgenic mice overexpressing aberrant human FUS[1-359] gene

Retinal damage is an optional sign in a number of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The aim of this work was to assess the structural and functional state of the retina in a murine model of ALS caused by overexpression of the aberrant FUS protein [1-359]. The retinal examination was carried out on 12 transgenic and 13 wild-type mice of 2.5–3 months of age. The study revealed not statistically significant higher level of ophthalmoscopic violations in FUS[1-359] mice. Moreover, gene expression assay confirmed an increased expression of the inflammatory genes Vegfa, Il1b, Il6, Icam1, Tnfa. However, despite the detected structural and functional abnormalities, western blot analysis and quantitative PCR did not detect the expression of the protein and mRNA products of the FUS transgene in the retina of FUS[1-359] mice.

Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis

Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.

Sequence determines the switch in the fibril forming regions in the low complexity FUS protein and its variants

Residues spanning different regions of the low-complexity domain of the RNA binding protein Fused in Sarcoma (FUS-LC) form two alternate fibril structures, with completely different core morphologies. Solid-state NMR experiments show that the 214 residue FUS-LC exclusively forms a fibril characterized by an S-bend (core-1 comprising of residues 39-95), while the rest of the protein is disordered. On the other hand, a C-terminal truncated variant (FUS-LC-C; residues 111-214) forms a fibril with a U-bend topology. The U-bend structure is not formed in FUS-LC, implying that both the fibril cores do not coexist. Using computer simulations based on a sequence-dependent coarse-grained SOP-IDP model, we show that these perplexing findings could be understood in terms of the population of fibril-like, N*, sparsely-populated excited states within the monomer conformation ensemble. The propensity to form core-1 is higher in FUS-LC as well as in several truncated variants. Interestingly, we find that sequence-dependent enthalpic effects, rather than conformational entropy of the disordered regions, determines the relative stabilities of the core-1 and core-2 topologies. Our simulations show that the core-2 structure can form only in truncated variants that do not contain the core-1 sequence.

ALS-linked FUS mutants affect the localization of U7 snRNP and replication-dependent histone gene expression in human cells

Genes encoding replication-dependent histones lack introns, and the mRNAs produced are a unique class of RNA polymerase II transcripts in eukaryotic cells that do not end in a polyadenylated tail. Mature mRNAs are thus formed by a single endonucleolytic cleavage that releases the pre-mRNA from the DNA and is the only processing event necessary. U7 snRNP is one of the key factors that determines the cleavage site within the 3ʹUTR of replication-dependent histone pre-mRNAs. We have previously showed that the FUS protein interacts with U7 snRNA/snRNP and regulates the expression of histone genes by stimulating transcription and 3ʹ end maturation. Mutations in the FUS gene first identified in patients with amyotrophic lateral sclerosis (ALS) lead to the accumulation of the FUS protein in cytoplasmic inclusions. Here, we report that mutations in FUS lead to disruption of the transcriptional activity of FUS and mislocalization of U7 snRNA/snRNP in cytoplasmic aggregates in cellular models and primary neurons. As a consequence, decreased transcriptional efficiency and aberrant 3ʹ end processing of histone pre-mRNAs were observed. This study highlights for the first time the deregulation of replication-dependent histone gene expression and its involvement in ALS.

Wild-type FUS corrects ALS-like disease induced by cytoplasmic mutant FUS through autoregulation

Mutations in FUS, an RNA-binding protein involved in multiple steps of RNA metabolism, are associated with the most severe forms of amyotrophic lateral sclerosis (ALS). Accumulation of cytoplasmic FUS is likely to be a major culprit in the toxicity of FUS mutations. Thus, preventing cytoplasmic mislocalization of the FUS protein may represent a valuable therapeutic strategy. FUS binds to its own pre-mRNA creating an autoregulatory loop efficiently buffering FUS excess through multiple proposed mechanisms including retention of introns 6 and/or 7. Here, we introduced a wild-type FUS gene allele, retaining all intronic sequences, in mice whose heterozygous or homozygous expression of a cytoplasmically retained FUS protein (FusΔNLS) was previously shown to provoke ALS-like disease or postnatal lethality, respectively. Wild-type FUS completely rescued the early lethality caused by the two FusΔNLS alleles, and improved age-dependent motor deficit and reduced lifespan associated with the heterozygous expression of FusΔNLS. Mechanistically, wild-type FUS decreased the load of cytoplasmic FUS, increased exon 7 skipping and retention of introns 6 and 7 in the endogenous mouse Fus mRNA, leading to decreased expression of the mutant mRNA. Thus, the wild-type FUS allele activates the homeostatic autoregulatory loop, maintaining constant FUS levels and decreasing the mutant protein in the cytoplasm. These results provide proof of concept that an autoregulatory competent wild-type FUS expression could protect against this devastating, currently intractable, neurodegenerative disease.