Monomethylated and unmethylated FUS exhibit increased binding to Transportin and distinguish FTLD-FUS from ALS-FUS
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Deposition of the nuclear DNA/RNA-binding protein Fused in sarcoma (FUS) in cytosolic inclusions is a common hallmark of some cases of frontotemporal lobar degeneration (FTLD-FUS) and amyotrophic lateral sclerosis (ALS-FUS). Whether both diseases also share common pathological mechanisms is currently unclear. Based on our previous finding that FUS deposits are hypomethylated in FTLD-FUS but not in ALS-FUS, we have now investigated whether genetic or pharmacological inactivation of Protein arginine methyltransferase 1 (PRMT1) activity results in unmethylated FUS or in alternatively methylated forms of FUS. To do so, we generated FUS-specific monoclonal antibodies that specifically recognize unmethylated arginine (UMA), monomethylated arginine (MMA) or asymmetrically dimethylated arginine (ADMA). Loss of PRMT1 indeed not only results in an increase of UMA FUS and a decrease of ADMA FUS, but also in a significant increase of MMA FUS. Compared to ADMA FUS, UMA and MMA FUS exhibit much higher binding affinities to Transportin-1, the nuclear import receptor of FUS, as measured by pull-down assays and isothermal titration calorimetry. Moreover, we show that MMA FUS occurs exclusively in FTLD-FUS, but not in ALS-FUS. Our findings therefore provide additional evidence that FTLD-FUS and ALS-FUS are caused by distinct disease mechanisms although both share FUS deposits as a common denominator.
KeywordsFrontotemporal lobar degeneration (FTLD) Amyotrophic lateral sclerosis (ALS) Fused in sarcoma (FUS) Arginine methylation Neurodegeneration Protein arginine methyltransferase 1 (PRMT1) Transportin-1
Fused in sarcoma (FUS) is a nuclear DNA/RNA-binding protein that is the pathological hallmark protein in abnormal cytoplasmic and nuclear inclusions in some cases of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) [24, 32, 35, 36, 54]. FTLD is the second most common early-onset dementia after Alzheimer’s disease (AD) and is clinically characterized by varying degrees of behavioural disturbances, personality changes and language impairment . The neuropathology of FTLD is classified according to the protein deposited in the central nervous system. FTLD cases show pathological deposition of either TAR DNA-binding protein of 43 kDa (TDP-43) , microtubule associated protein tau (Tau)  or Fused in sarcoma (FUS) [32, 35, 36], hence they are classified as FTLD-TDP, FTLD-Tau or FTLD-FUS . Only very rare FTLD cases cannot be classified in these categories, including familial FTLD linked to chromosome 3 (FTD-3) [17, 18] (caused by mutations in the CHMP2B gene) and FTLD cases with no inclusions (FTLD-ni) . FTLD-FUS represents about 10 % of all FTLD cases  and comprises three distinct pathological subtypes, namely atypical FTLD-U (aFTLD-U) , neuronal intermediate filament inclusion disease (NIFID)  and basophilic inclusion body disease (BIBD) . Genetic and neuropathological analyses revealed overlapping disease characteristics of ALS and FTLD . Although TDP-43 is the most common protein underlying ALS pathology, some rare ALS cases are associated with FUS mutations and show instead FUS aggregates (ALS-FUS) [24, 54].
Arginine methylation is mediated by protein N-arginine methyltransferases (PRMTs), which catalyse methyl group addition in two sequential steps (Fig. 1b). First, addition of a single methyl group generates monomethylated arginine (MMA). Subsequent addition of a second methyl group leads to a dimethylated form that can occur either as ADMA or symmetric N,N-dimethylarginine (SDMA) [2, 57]. In mammalian cells, there are nine PRMTs that all catalyse the first step that generates the MMA form, but different PRMTs are responsible for the second step (Fig. 1b). Type I PRMTs (which include PRMT1, PRMT2, PRMT3, PRMT4/CARM1, PRMT6 and PRMT8) mediate asymmetric arginine dimethylation. PRMT1 is the main type I PRMT and is responsible for 90 % of all ADMA enzyme activity in mammalian cells . In contrast, type II PRMTs (PRMT5 and PRMT9) are responsible for SDMA [2, 57]. Finally, it has been described that PRMT7 exclusively catalyses the generation of MMA and is accordingly classified as a type III PRMT . The MMA form has long been considered to be an intermediate form between the unmethylated arginine (UMA) and ADMA/SDMA forms. However, recently it has been shown that levels of MMA proteins increase after loss of PRMT1  and that the increase of MMA is not coupled with a subsequent increase in ADMA modifications . These findings suggest that MMA is not just an intermediate form, but may have selective biological functions. PRMTs mostly target arginines in regions rich in glycines and arginines . Proteins of the FET family (FUS, EWS and TAF-15) contain three arginine-glycine-glycine (RGG) domains that are thought to mainly undergo asymmetric dimethylation by PRMT1 [1, 4, 19, 21, 43, 46], i.e. all three proteins appear to be present mainly in the ADMA form under physiological conditions. Some MMA sites have also been reported for the FET proteins [16, 48], whereas there is no evidence of symmetric dimethylation in FET proteins .
In the present study, we aimed at characterizing the methylation pattern of FUS in the pathological inclusions in brains of FTLD-FUS patients. To do so, we developed novel monoclonal antibodies specific for the UMA or MMA RGG3 domain of FUS. By using these antibodies along with a previously developed antibody specific to the ADMA RGG3 domain of FUS , we show that genetic or pharmacological inhibition of PRMT1 not only results in UMA FUS, but also in alternatively methylated MMA FUS. Moreover, we demonstrate that MMA FUS exhibits increased binding affinities to Transportin-1 similar to UMA FUS, whereas ADMA FUS only binds very weakly to Transportin-1. Finally, we provide evidence that FUS inclusions in FTLD-FUS contain UMA and, to a lesser extent, MMA FUS, in contrast to ALS-FUS where FUS appears to be exclusively asymmetrically dimethylated.
Materials and methods
Cell lines, cell culture, transfections and inhibitor treatment
Human cervical carcinoma cells (HeLa) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % (v/v) fetal calf serum (FCS), glutamine and penicillin/streptomycin at 37 °C with 5 % CO2 in a tissue culture incubator.
Mouse embryonic stem (mES) cells, PRMT1 knockout (−/−) and wild-type controls (+/+) , were cultured with STEMPAN GMEM (Cat. No. P08-50600, PAN Biotech) supplemented with 10 % (v/v) fetal bovine serum (PANsera Cat. No. P30-2602, PAN Biotech), penicillin/streptomycin and leukaemia inhibitory factor (ESGRO Mouse LIF Medium Supplement, Cat. No. ESG1107, Millipore) at a final concentration of 103 U/ml. Adenosine-2′,3′-dialdehyde (AdOx; Sigma) was used as a methyltransferase inhibitor  on HeLa cells for 48 h at a concentration of 20 μM. AdOx treatment has been used to inhibit dimethylation of proteins  and to increase not only the UMA but also the MMA forms . FUS, EWS and TAF-15 knockdowns were achieved by using the ON-TARGET plus FUS SMARTpool L-009497, the ON-TARGET plus EWSR1 SMARTpool L-005119 and the ON-TARGET plus TAF-15 SMARTpool L-008930, respectively, all from Dharmacon. A non-targeting siRNA (ON-TARGET plus NT siRNA #3, D-001810-03 from Dharmacon) was used as a negative control. siRNAs were delivered to cells by reversed transfection using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The effect of the knockdown was assessed 48 h post-transfection.
Generation of monoclonal antibodies and peptides
Monoclonal antibodies were generated in LOU/C rats or C57BL/6 mice against ovalbumin-conjugated FUS473–503 peptide (RGGRGGYDRGGYRGRGGDRGGFRGGRGGGDR), corresponding to the RGG3 domain of FUS, in which all nine arginines were either unmethylated (UMA FUS) or monomethylated (MMA FUS) (Fig. 1a). The ADMA FUS antibody (9G6) was previously described and characterized . Rat monoclonal antibodies were also raised against the ADMA RGG3 domain of EWS (EWS564–583 GRGGPGGMRGGRGGLMDRGG) and TAF-15 (TAF-15458–477 DRGGGYGGDRGGGYGGDRGG), using ovalbumin-conjugated peptides. All antibodies were produced by standard procedures.
Peptides were synthesized by Peps4LS GmbH, or Peptide Specialty Laboratories GmbH. For the methylated peptides, modified amino acids (ADMA or MMA, respectively) were used instead of arginine during synthesis. Peptides were purified by gradient HPLC-purification on a C-18 preparative HPLC column and the single fractions were characterized by UV trace of an analytical HPLC spectrum. Only fractions with a >90 % purity were combined and confirmed by MALDI mass spectrometry.
The hybridoma supernatants were first screened by ELISA using the corresponding ADMA, MMA or UMA peptides as antigens. Next, they were assessed in immunoblotting of cell culture lysates of HeLa cells. The UMA FUS-specific clone 14G1 (Rat IgG 2a) showed the best performance, for MMA FUS-specificity, clone 15E11 (Rat IgG 2a) was chosen. To confirm the results obtained with these antibodies, we generated and selected an additional monoclonal UMA FUS-specific antibody, (2A3; Mouse IgG 2b), and an additional monoclonal MMA FUS-specific antibody (18E11; Rat IgG 2a). Similarly, we selected the best ADMA EWS-specific clone (21B1; Rat IgG1) and the best ADMA TAF-15-specific clone (Rat 12F11; Rat IgG 2c). All hybridomas were subcloned at least twice to ensure monoclonality and stability of the clones. Where necessary, antibodies were purified via protein G column.
The following commercial antibodies were used: EWS-specific mouse monoclonal antibody G5 sc28327 (Santa Cruz) and rabbit polyclonal antibody H-60 sc28865 (Santa Cruz); FUS-specific mouse monoclonal antibody 4H11 (Santa Cruz) and rabbit polyclonal A300-294A (Bethyl) and HPA008784 (Sigma); HA-specific mouse monoclonal antibody HA.11 (Covance); MonomethylArginine (R*GG)-specific rabbit monoclonal antibody D5A12 #8711 (Cell Signalling); MonomethylArginine-specific monoclonal rabbit antibody Me-R4-100 #8015 (Cell Signalling); PRMT1-specific rabbit monoclonal EPR3292 (Abcam); TAF-15-specific rabbit antibody TAF15-308A (Bethyl), rabbit polyclonal antibody SAB2102361 (Sigma) and mouse monoclonal antibody H00008148 (Abnova); Transportin-specific rabbit antibody clone D45 (Sigma); α-Tubulin-specific mouse monoclonal antibody clone B-5-1-2 (Sigma).
The secondary antibodies for immunoblotting were horseradish peroxidase (HRP)-conjugated goat anti-mouse, anti-rabbit or anti-rat IgG (Promega) or mouse anti-rat IgG2a, IgG2b and IgG2c (home made).
Total cell lysates were prepared in ice-cold RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulphate) freshly supplemented with Protease inhibitor Cocktail (Roche), sonicated (Bioruptor from Diagenode), and their protein concentration was determined by BCA protein assay (Interchim). Lysates were then supplemented with SDS–PAGE sample buffer (62.5 mM Tris–HCl pH 6.8, 2 % sodium dodecylsulfate, 0.03 % bromophenol blue, 143 mM β-mercaptoethanol, 10 % glycerol) and boiled for 5 min.
For immunoprecipitation, whole mES cell extracts were prepared in mild lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 % Triton X-100) for the immunoprecipitation of FUS, EWS and MMA proteins, or in RIPA buffer for the immunoprecipitation of TAF-15. The lysates were pre-cleared with washed Protein G Sepharose beads (Thermo Scientific) for 30 min at 4 °C, followed by incubation for 2 h at 4 °C with the antibody of interest coupled to Protein G beads. The beads were washed three times with the lysis buffer and boiled in 2× SDS-PAGE sample buffer to elute bound proteins.
Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Imobilon-P, Millipore). Membranes were blocked in 5 % powdered milk in TBST and incubated in primary antibody overnight at 4 °C. Membranes were probed with HRP-coupled secondary antibodies and developed with the chemiluminescence detection reagents ECL or ECL plus (both from Thermo Scientific).
To examine solubility of FET proteins, a sequential extraction was performed as described  with some modifications. Briefly, cells were washed with PBS, lysed in cold RIPA buffer and sonicated. Lysates were centrifuged at 180,000g for 30 min at 4 °C. After removal of the RIPA-soluble fraction, RIPA-insoluble pellets were washed three times with RIPA buffer, sonicated and re-pelleted. Insoluble pellets were dissolved in urea buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 30 mM Tris, pH 8.5). Both soluble and insoluble fractions were supplemented with SDS-PAGE sample buffer and boiled.
Neuronal culture, transfection and treatment
Cortical neurons were prepared from embryonic day 18 Sprague–Dawley rat embryos and cultivated in Neurobasal medium (ThermoFisher) supplemented with 2 % B27 (ThermoFisher), 1 % penicillin/streptomycin, 0.25 % glutamine and 0.125 % glutamate as described previously . Cells were harvested 7 days later (DIV7) in RIPA buffer. Adox (10 µM) treatment was started 48 h prior to harvesting.
In vitro pull-down assay
In vitro pull-down assays were performed as previously described . Briefly, N-terminally biotinylated UMA-FUS473–503, MMA-FUS473–503 and ADMA-FUS473–503 peptides were immobilized on streptavidin Sepharose beads (GE Healthcare) and blocked in wash buffer (20 mM sodium phosphate buffer pH 7.4, 150 mM KCl, 0.5 mM EDTA, 5 mM MgCl2, 10 % glycerol, 1 mM DTT) supplemented with 0.5 mg/ml BSA. The peptide-coupled beads were then incubated with increasing amounts of recombinant TRN–His6 or His6–GST for 1–3 h at 4 °C. The beads were washed, boiled in SDS-PAGE sample buffer and eluted proteins were resolved by SDS-PAGE and visualized by staining with GelCode Blue Stain Reagent (Thermo Scientific).
Protein expression and purification
Escherichia coli (E. coli) BL21 (DE3) cells were transformed with a modified version of the pETM11 expression vector (including a His6, protein A tag and a tobacco etch virus–TEV- protease cleavage site) harbouring the E. coli codon optimize gene of Transportin-1. One litre of M9 minimal medium was inoculated, cultures were grown for 2 days at 25 °C, diluted to OD 1.0 and induced with 0.5 mM IPTG for either 6 h at 25 °C or 14 h at 19 °C. Cells were resuspended in 30 ml purification buffer [110 mM potassium acetate, 20 mM Hepes, pH 8.0, 2 mM MgCl2, 2 mM β-mercaptoethanol (BME), 5 % (v/v) glycerol and 20 mM Imidazole], sonicated and applied to a Ni–NTA agarose column according to the manufacturers instruction. The protein was eluted with the same buffer including 200 mM imidazole and further purified via size exclusion chromatography on an ÄKTA pure system equipped with HiLoad 16/600 Superdex 200 pg (for Transportin-1; column GE Healthcare) using purification buffer. The protein-containing fractions were merged and incubated with 0.2 mg TEV protease overnight at 4 °C. On the next day, the protein solution was applied on the Ni–NTA agarose column to remove the tag and the His6-tagged TEV protease. Protein was freshly prepared for ITC runs, stored at 4 °C, and buffer exchanged prior the measurements on an ÄKTA pure system equipped with a HiPrep (26/10) desalting column (GE Healthcare) pre-equilibrated with freshly prepared ITC interaction buffer (50 mM Tris, 150 mM NaCl, 2 mM tris(2-carboxyethyl)phosphine, pH 6.7). Protein solution was concentrated using Amicon Ultra-15 (Millipore, 3 kDa molecular weight cut-off) centrifugal filter units to the desired concentrations.
Isothermal titration calorimetry (ITC)
Binding affinities of FUS RGG3 peptides to Transportin-1 were determined using a VP-ITC Microcal calorimeter (Microcal, Northhampton, USA) at 25 °C with a stirring speed of 300 rpm. Titrations employed an initial delay of 300 s, a 9.6 µl initial injection volume followed by 300 s delay, and 35 × 19.6 µl injections of FUS peptides into the Transportin-1 solution. Concentrations of Transportin-1 were 10 µM for the UMA and MMA RGG peptides and 15 µM for the ADMA peptide, respectively. Concentrations of UMA RGG peptide were 100, 120 µM for MMA RGG peptide and 150 µM for the ADMA peptide. The ITC datasets were analysed with the program MicroCal Origin software version 7.0 assuming a single binding site.
Immunohistochemistry and immunofluorescence on human post mortem tissue
Human post mortem central nervous system (CNS) tissue was provided by the Neurobiobank Munich, Ludwig-Maximilians-University (Munich, Germany) and the Brain Banks affiliated with the University of Tübingen, Germany, University of British Columbia, Canada, and University of Oxford, United Kingdom. Consent for autopsy and research was obtained from the legal representative in accordance with local institutional ethical review committees.
Studied FUS-opathy cases with robust pathology in selected neuroanatomical regions included aFTLD-U (n = 4), BIBD (n = 2), NIFID (n = 3) and four ALS-FUS cases with three different FUS mutations, p.R521C (n = 2), p.P525L (n = 1), and p.R514S/E516 V (n = 1).
Immunohistochemistry was performed on ~5 µm thick formalin fixed paraffin embedded sections using the Ventana BenchMark XT automated staining system (Ventana, Tuscon, AZ) with the iVIEW DAB detection kit. Boiling of sections in citrate buffer (pH 6) for 20 min was performed as antigen retrieval for all stainings and incubation of primary antibodies was performed overnight at 4 °C. For double-label immunofluorescence, the secondary antibodies were Alexa Fluor 594 conjugated anti-rabbit and or Alexa Fluor 488 conjugated anti-rat IgG (Invitrogen, 1:500). Hoechst 33342 or DAPI was used for nuclear counterstaining. Sections were treated with Sudan black to reduce autofluorescence.
Generation and characterization of UMA FUS- and MMA FUS-specific antibodies
To characterize the methylation patterns of FUS in post mortem brains of FTLD and ALS patients we developed highly selective monoclonal antibodies against the differentially methylated RGG3 domain of FUS (peptide epitope FUS473–503; Fig. 1a). This domain is localized next to the PY-NLS and contains nine arginine residues that can be asymmetrically dimethylated [40, 46]. UMA FUS473–503 and MMA FUS473–503 specific antibodies were newly developed for this study, whereas the ADMA FUS-specific antibody 9G6 has been previously developed and characterized  (Fig. 1a). Hybridoma supernatants were screened by ELISA against all peptides, and those with a strong selective signal but no significant cross-reactivity to the other peptides were further tested by immunoblotting on HeLa cells lysates. To validate specificity, we performed siRNA-mediated FUS knockdowns and treated cells with the methylation inhibitor AdOx (adenosine-2′-3′-dialdehyde). AdOx is a global methyltransferase inhibitor that reduces the ADMA of proteins  and conversely increases the UMA but also the MMA forms . After screening a large number of antibodies, we selected an UMA FUS (clone 14G1) and MMA FUS (clone 15E11) antibody that recognized a specific band in AdOx-treated HeLa cells that was selectively absent upon knockdown of FUS but not of the other FET family members TAF15 or EWS (Fig. 1c). We also used our previously described ADMA FUS-specific antibody (clone 9G6) , which shows a signal in untreated cells that disappears upon treatment with AdOx or knockdown of FUS (Fig. 1c).
Genetic and pharmacological reduction of PRMT1 results in a switch from asymmetrically dimethylated FUS to unmethylated and monomethylated FUS
To exclude cross-reactivity of our antibodies against other methylation patterns in PRMT1 knockout cells, we performed an antigen absorption assay with the three different peptides used for immunization (Supplementary Fig. 1a, b). The specific band recognized by the 14G1 UMA FUS antibody selectively disappeared upon preincubation of the antibody with the UMA FUS473–503 peptide, but not with the MMA FUS473–503 or the ADMA FUS473–503 peptide (Supplementary Fig. 1a). Likewise, the signal obtained with the 15E11 MMA FUS antibody selectively disappeared upon preincubation of the antibody with the MMA FUS473–503 peptide but not with the others (Supplementary Fig. 1b). These results confirm the selectivity of the 14G1 and 15E11 antibodies and, importantly, demonstrate the lack of cross-reaction with the other methylation patterns.
Loss of PRMT1 increases monomethylation of all members of the FET protein family, but does not affect their solubility
Unmethylated and monomethylated FUS have a higher affinity for Transportin-1
TRN binding to FUS. dissociation constants (k D) as determined by isothermal titration calorimetry
k D (µM)
0.7 ± 0.4
4.5 ± 0.9
12.4 ± 10.7
Inclusions in FTLD-FUS but not ALS-FUS patients contain unmethylated and monomethylated FUS
Taken together, we demonstrate that the inclusions are clearly labelled with both the UMA FUS and the MMA FUS antibodies, which further supports our hypothesis of impaired FUS methylation regulation in FTLD-FUS pathogenesis.
Comparison of FTLD-FUS and ALS-FUS pathology
Two principal types of posttranslational modifications (PTMs) have been described for the FET proteins: arginine methylation and phosphorylation [8, 15, 23, 26]. Both methylation and phosphorylation can affect the nuclear-cytoplasmatic localization of FET proteins [3, 7, 13, 14, 28, 52] and they may also be involved in the regulation of RNA/DNA binding, protein–protein interaction , stress granule recruitment  or the solubility of the FET proteins [42, 45, 58]. Furthermore, it has recently been described that phosphorylation of the N-terminus of FUS mediates its cytoplasmic translocation after DNA damage .
The hypothesis that FUS could be hypomethylated in FTLD-FUS emerged from our previous finding with an antibody specific for ADMA FUS. We could show that inclusions in ALS-FUS patients were labelled with this antibody, but inclusions in FTLD-FUS cases were not . This implied that inclusions in FTLD-FUS do not contain ADMA FUS. However, the experiments did not exclude that the absent staining was caused by different conformations of FUS in inclusions in FTLD-FUS, with the epitope for our ADMA FUS antibody being inaccessible.
To model the hypomethylated state that we hypothesise to occur in FTLD-FUS, we used mES cells lacking PRMT1 . Using this cellular model, we investigated how the pattern of methylation of FUS and the other FET proteins changes when FET proteins are not properly methylated by PRMT1. As expected, we observed a decrease in ADMA FUS, EWS and TAF-15 and an increase in UMA FUS upon PRMT1 knockout. Remarkably, we also observed an increase in MMA FET proteins. Thus, all FET proteins adopt differentially methylated forms (UMA and MMA) upon loss of PRMT1 activity.
Little is known about the role of MMA as a regulator of protein function but, interestingly, several other RNA-binding proteins besides FUS can be monomethylated [16, 48], which implies that this PTM probably plays a pivotal role in RNA-binding protein regulation. Previous studies identified rare MMA sites in EWS [4, 43] and, more recently, two reports independently identified proteins that contain MMA in human cell lines through a proteomic approach [16, 48]. Both studies applied an immunoaffinity purification of proteins containing MMA, followed by identification of these proteins by mass spectrometry [16, 48]. The most common MMA-containing proteins identified were those involved in RNA processing and transcriptional regulation. Strikingly, among the MMA proteins all three FET proteins were found. Interestingly, Guo et al.  also describes a tissue-specific distribution of MMA proteins and report that in mouse brains most MMA proteins are involved in synaptic transmission. Whether MMA sites are merely a transition state between UMA and ADMA or indeed a distinct posttranslational modification with its own biological function is still unknown. This latter hypothesis is supported by the fact that there is a methyltransferase, PRMT7, that only catalyzes monomethylation . Moreover, it has been suggested that a still uncharacterized MMA-specific demethylase may exist . Monomethylation of lysines is already known to have its own biological function, namely labelling proteins for degradation through the methyl degron .
A still unanswered question is how the hypomethylated state may arise in brains of FTLD-FUS patients. One possible explanation would be a deficiency in PRMT activity, but this hypothesis seems unlikely, given that these enzymes methylate a broad range of substrates besides FET proteins, that are not deposited in FTLD-FUS . Furthermore, no mutations in PRMT genes have been found in FTLD-FUS cases . However, only three PRMTs have been investigated (PRMT1, PRMT3 and PRMT8), hence the remaining PRMTs and other genes involved in arginine methylation of FET proteins should still be considered as candidates for genetic studies in FTLD-FUS.
Despite the fact that some studies have reported that hypomethylation favours protein aggregation [42, 45, 58], our data do not support the idea that the hypomethylation of FET proteins makes them more prone to aggregate. In contrast, our present study strengthens the hypothesis that deposition of the FET proteins in FTLD-FUS may be caused/favoured by the higher affinity of both UMA and MMA FUS to Transportin-1. Thus, we speculate that UMA and MMA FET proteins present in FTLD-FUS may lead to irreversible FET-Transportin-1 binding and eventually co-deposition of these proteins in cytoplasmic and intranuclear neuronal and glial inclusions. However, alternative mechanisms (such as phosphorylation of FUS) may contribute to FUS redistribution and its subsequent pathological deposition in FTLD-FUS .
In conclusion, this study describes the first PTM specifically associated with FTLD-FUS, namely MMA FUS. We demonstrate that MMA FUS and UMA FUS deposition are pathological hallmarks of FTLD-FUS that are not found in ALS-FUS caused by FUS mutations. This reinforces the idea that the two diseases are most likely caused by different pathomechanisms (Table 2)  and that loss of proper arginine methylation of the FET proteins may be involved in FTLD-FUS pathology. Furthermore, we highlight the fact that FET proteins are not only dimethylated or unmethylated, but there is a novel so far overlooked posttranslational modification of FUS (MMA FUS), whose exact biological and pathological role needs to be elucidated in the future.
We thank Alice Suelzen for technical assistance. We thank H. Earl Ruley (Vanderbilt University School of Medicine; Nashville, TN, USA) for kind gift of mouse embryonic stem cells (mES) (PRMT1 knock-out and wild-type controls) and Elmar Wahle (Martin-Luther-Universität Halle-Wittenberg, Germany) for gift of reagents.
This work was supported by the European Research Council under the European Union’s Seventh Framework Program (FP7/2007–2013)/ERC Grant Agreement No. 321366-Amyloid (advanced grant to C.H.), the Deutsche Forschungsgemeinschaft (German Research Foundation) within the framework of the Munich Cluster for Systems Neurology (EXC 1010 SyNergy D.D., C.H.) and the Emmy Noether program DO 1804/1-1 (to D.D.), and the general legacy of Mrs. Ammer (to the Ludwig-Maximilians-University/the chair of C.H.) and the German Helmholtz Association (Grant VH-VI-510 to C.H. and M.N.; Grant W2/W3-036 to M.N.). M.S. was supported by a grant from the Fondo de Investigación Sanitaria (FI09/00732), Instituto Carlos III, Madrid, Spain. T.M. was supported by the Bavarian Ministry of Sciences, Research and the Arts (Bavarian Molecular Biosystems Research Network), the German Research Foundation (Emmy Noether program MA 5703/1-1), the Centre for Integrated Protein Science Munich (CIPSM), the President’s International Fellowship Initiative of CAS (No:2015VBB045), and the National Natural Science Foundation of China (No. 31450110423). We gratefully acknowledge the support of the NOMIS foundation (to M.D.R.), the Holcim Stiftung zur Föderung der wissenschaftlichen Fortbildung (to M.D.R.), and the Fondation Dufloteau (to M.D.R.).
Compliances with ethical standards
Conflict of interest
The authors disclose no conflicts of interest.
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