FUS gene is dual-coding with both proteins united in FUS-

6 1 Department of Biochemistry and Functional Genomics, Université de Sherbrooke, Sherbrooke, 7 Québec, Canada; 8 2 PROTEO, Quebec Network for Research on Protein Function, Structure, and Engineering; 9 3 Immunology and Cell Biology Department, Université de Sherbrooke, Sherbrooke, Québec, 10 Canada; 11 4 The Francis Crick Institute, 1 Midland Road, London, UK; 12 5 Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, Queen 13 Square, London, UK; 14 6 Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, 15 Canada; 16 7 Division of Neurology, Department of Medicine, Sunnybrook Health Sciences Centre, University 17 of Toronto, Toronto, Canada. 18


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food. The treatment induces a conformational change in the Elav-GeneSwitch driver, which allows 244 activation of the UAS promoter and thus expression of the target protein. We retrieved flies at 245 selected time points to validate protein expression in the RU-486 treated population through 246 time, while the controls showed no expression (Fig 5B). 247 The motor neuron degeneration linked to ALS provokes a progressive locomotion loss measurable 248 with a well-described climbing assay 52 . The control populations did not show any significant 249 locomotion loss at day 1, 10 nor 20 (Fig 5C-E). Similarly to the RU-486 treated control group did 250 not show a significant effect, although a decrease in climbing ability could be observed as 251 previously reported 53,54 (mCherry transgenic flies - Fig 5C). AltFUS flies did not show any 252 significant locomotion loss through time (Fig 5C). This result is consistent with the in cellulo data 253 showing altFUS alone is not sufficient to provoke pathological hallmarks. As previously shown with 254 this model 8 , the bicistronic FUS flies, which express both FUS and altFUS proteins, displayed a 255 significant locomotion loss (Fig 5D). Bicistronic ALS-linked FUS-R495x flies showed an even greater 256 motor neuron degeneration through time compared to FUS (Fig 5E). Monocistronic FUS (Ø) (Fig 5D)  257 and FUS (Ø) -R495x (Fig 5E)  As of today, over 50 mutations in the FUS gene have been associated with ALS 1 . However, most 265 of these locate at the carboxyl end of the protein and as such have no effect on altFUS (Fig 1A). 266 25 quenched using 1 % formic acid and supernatant was collected. Beads were washed once with 524 acetonitrile/water/formic acid (1/1/0.01 v/v) and pooled with supernatant. Peptides were dried 525 with a speedvac, desalted using a C18 Zip-Tip (Millipore Sigma, Etobicoke, Ontario, Canada) and 526 resuspended into 30 μl of 1% formic acid in water prior to MS analysis. 527

Mass-spectrometry analysis 528
Peptides were separated in a PepMap C18 nano column (75 μm × 50 cm, Thermo Fisher Scientific). 529 The setup used a 0-35% gradient (0-215 min) of 90% acetonitrile, 0.1% formic acid at a flow rate 530 of 200 nL/min followed by acetonitrile wash and column re-equilibration for a total gradient 531 duration of 4 h with a RSLC Ultimate 3000 (Thermo Fisher Scientific, Dionex). Peptides were 532 sprayed using an EASYSpray source (Thermo Fisher Scientific) at 2 kV coupled to a quadrupole-533 Orbitrap (QExactive, Thermo Fisher Scientific) mass spectrometer. Full-MS spectra within a m/z 534 350-1600 mass range at 70,000 resolution were acquired with an automatic gain control (AGC) 535 target of 1e6 and a maximum accumulation time (maximum IT) of 20 ms. Fragmentation (MS/MS) 536 of the top ten ions detected in the Full-MS scan at 17,500 resolution, AGC target of 5e5, a 537 maximum IT of 60 ms with a fixed first mass of 50 within a 3 m/z isolation window at a normalized 538 collision energy (NCE) of 25. Dynamic exclusion was set to 40 s. Mass spectrometry RAW files were 539 searched with Andromeda search engine implemented in MaxQuant 1.5.5.1. The digestion mode 540 was set at Trypsin/P with a maximum of two missed cleavages per peptides. Oxidation of 541 methionine and acetylation of N-terminal were set as variable modifications, and 542 carbamidomethylation of cysteine was set as fixed modification. Precursor and fragment 543 tolerances were set at 4.5 and 20 ppm respectively. Files were searched using a target-decoy 544 approach against UniprotKB (Homo sapiens 03/2017 release) with the addition of altFUS 545 sequence for a total of 92,949 entries. The false discovery rate (FDR) was set at 1% for peptide-546 spectrum-match, peptide and protein levels. Protein interactions were then scored using the 547 26 SAINT algorithm, with Mock cells as control and the magnetic FLAG beads in HEK293 cells 548 crapome 66 . Proteins with a SAINT score above 0.99 were considered, as well as those presenting 549 a SAINT score above 0.88 with a minimum of two unique peptides. 550

Biological processes and cellular compartment enrichment analysis 551
Proteins identified in altFUS interactome were screened for cellular compartment and biological 552 processes enrichment using Gene Ontology (GO) enrichment. Proteins were queried against the 553 whole Human Proteome for cellular compartment and against the Human mitochondrial 554 proteome (MitoCarta 2.0) for biological processes. The statistical analysis used a Fisher's Exact 555 test with a FDR set at 1 %. 556

Autophagic flux measurements 557
The mCherry-GFP-LC3 was used to evaluate the autophagic vesicles within HeLa cells by confocal 558 microscopy. Before fusion with the lysosome, the LC3 molecules on the autophagosome display 559 a yellow fluorescence (combined mCherry and GFP fluorescence). After fusion, the GFP 560 fluorescence is quenched by the lysosomal pH, and as such the LC3 molecules display a red signal 561 (mCherry alone). This allows a visual representation of the autophagic flux in a given cell. Cells 562 treated with 50 nM Bafilomycin for 4 hours were used as a positive control to validate each 563 independent experiment. Observations were made across 2 technical duplicates for each 564 biological condition, across 3 independent experiments (n=3). Alternatively, the autophagic flux 565 was also evaluated by LC3 probing before and after bafilomycin treatment (50 nM for 4 hours). 566 The quantification corresponds to the treated / untreated ratio of LC3-II abundance. 567

Cytoplasmic aggregates measurements 568
Images of HeLa cells were taken by confocal microscopy and then processed using the Image J 3D 569 Objects Counter plugin. FUS cytoplasmic aggregates were then quantified in number and size 570 27 (μm 2 ) for each cell. A total of 100 cells across two technical replicates were taken for each 571 independent experiment (n=3, i.e. a minimum of 300 cells per biological conditions). 572 Transgenic Drosophila and climbing assay 573 The bicistronic constructs, FUS and FUS-R495x, and the monocistronic constructs, altFUS, FUS (Ø) 574 and FUS (Ø) -R495x, were subcloned in the pUASTattB expression vector for site specific insertion 575 into attP2 on chromosome 3. Transgenic flies were generated by Best Gene (Best Gene Inc., 576 California, USA). The Elav-GeneSwitch-GAL4 driver ( background and were cultured on standard medium at 25°C or room temperature. Transgenic 581 flies were crossed with the Elav-GeneSwitch-GAL4 driver strain. The F1 was equally divided in two 582 groups with equal proportion of males and females: one group will feed on standard food 583 supplemented with ethanol (0.2 % -control flies), the other on standard food supplemented with 584 RU-486 at 10 μM diluted in ethanol (induced flies). The climbing assay was performed as 585 previously described 52 . Briefly, flies were transferred into an empty vial and tapped to the bottom. 586 After 18 s, the number of flies at the top of the tube were considered successful. The assay was 587 done at day 1, 10 and 20 post-induction, across 4 independent F1. Five flies were taken at day 1, 588 10 and 20 post-induction to validate expression of the proteins of interest. 589

Statistical analyses and representation 590
Unless otherwise stated, the statistical analysis carried was a two-way ANOVA with Tukey's 591 multiple comparison correction. The box plots represent the mean with the 5 to 95 % percentile. 592 The bar graphs represent the mean, and error bars correspond to the standard deviation. When 593 28 using parametric tests, normality of data distribution was verified beforehand using the Shapiro-594 the beginning of the FUS protein (green) and the whole altFUS protein (red). In F, the 'Peptide' 797 track contains all the peptides identified by the OpenProt resource using the classical spectrum-798 centric approach. The peptides sequences are indicated and are unique to altFUS or its isoform 799 (see Fig EV2B for an example spectra). In G, the 'Peptide' track contains all the peptides identified 800 by our peptide-centric approach. The peptides indicated matched better to at least one spectra 801 than any known protein, and are coloured in yellow if they matched better than any known 802 protein with any PTM (see Fig EV2C for an example spectra). The peptides sequences are indicated 803 and are unique to altFUS or its isoform. 804 identified by AP-MS (see Fig EV5) indicating their enrichment (Fold-change over control) and their 853 SAINT probability score. Proteins above the 0.8 threshold are indicated in black, others in grey. 854 AltFUS is indicated in red (bait) and preys known to regulate the autophagy or the cellular stress 855 39 response are indicated in green. F, Subcellular localizations of proteins identified by AP-MS from 856 (see Fig EV5). G, Enrichment of biological processes in altFUS interacting proteins compared to 857 the Human mitochondrial proteome (n=2, Fisher's Exact test with FDR < 0.1 %). The number of 858 proteins identified in each GO term is indicated next to the corresponding bar. 859 for FUS (ENSP00000254108) and its isoform (ENSP00000369594) in panel D (blue) and altFUS 923 (IP_243680) and its isoform (IP_243691) in panel E (green). The residues are coloured based on 924 their degree of identity. F, Heatmap of altFUS protein sequences identity across 84 species (see 925 Appendix Table 2 and Source Data 1). Primates, Rodents and Mammals in general display strong 926 protein conservation. The sequence identity is colored from blue (0 %) to red (100 %). 927