Fig 2: NOVA1 expression in postmortem MN in lumbar spinal cord from ALS and Ctrl a Schematic illustrating the basic analysis strategy for NOVA1 expression in human tissue. We performed IF staining, spinning disc confocal microscopy and subsequent analysis of single MNs for NOVA1 mean fluorescent intensity and TDP-43 pathology in n = 7 Ctrl and n = 9 sALS. b Representative images of MN with nuclear TDP-43 (no TDP-43 pathology, purple arrow) and without nuclear TDP-43 (with TDP-43 pathology, red arrow) in sALS patients. Pictures stained for TDP-43 (green), NOVA1 (red), bIII-tubulin (gray) and DAPI (blue). Nuclear (cyan), cytoplasmic (fuchsia) and lipofuscin (yellow) areas analyzed are surrounded. Scale bars: 25µm. c Scatter plot of mean nuclear NOVA1 staining intensities in individual MNs from different ALS patients without (purple) and with (red) TDP-43 pathology. Horizontal black lines represent medians. Statistical analysis was performed using a mixed effects model. d Scatter plot of mean cytoplasmic NOVA1 intensity in individual MNs from different ALS patients without (purple) and with (red) TDP-43 pathology. Horizontal black lines represent medians. Statistical analysis was performed using a mixed effects model. e Representative immunofluorescence images of MNs in lumbar spinal cord from control individuals (black) and from sALS patients without (purple) and with TDP-43 pathology (red). Sections were stained for TDP-43 (green), NOVA1 (red), bIII-tubulin (gray) and DAPI (blue). Nuclear (cyan) and cytoplasmic (fuchsia) areas analyzed are surrounded. Blue arrowheads indicate cytoplasmic NOVA1 accumulations. Scale bar: 25µm. f Scatter plot of bIII-tubulin normalized mean nuclear NOVA1 staining intensities of single MNs (dots) their average in an individual (line). Dashed line represents median in Ctrl. Statistical significance was determined by nested one-way ANOVA. *P value < 0.05; **P value < 0.01; ***P value < 0.001; ****P value < 0.0001. Significant variation between individuals within a group: P value = 0.0001. g Scatter plot of bIII-tubulin normalized mean cytoplasmic NOVA1 intensities of single MN (dots) their average in an individual (line). Dashed line represents median in Ctrl. Statistical significance was determined by nested one-way ANOVA. *P value < 0.05; **P value < 0.01; ***P value < 0.001; ****P value < 0.0001. Significant variation between individuals within a group: P value > 0.05. h Scatter plot illustrating NOVA1 expression converted into expression Z-scores based on control means from postmortem ALS LCM datasets from [32] and [55] in Ctrl (black), sALS samples with regular STMN2 levels (STMN2 expression Z-score > −2, purple) and sALS samples with reduced STMN2 levels (STMN2 expression Z-score < −2, red). Black horizontal lines indicate median and interquartile ranges. Statistical significance was determined by Kruskal-Wallis test and Dunn's post hoc test to identify changes between individual groups. *P value < 0.05; **P value < 0.01; ***P value < 0.001; ****P value < 0.0001 i Scatter plot illustrating NOVA1 expression converted into expression Z-scores based on Ctrl means from postmortem ALS motor cortex datasets from UCSD and NYGC cohorts [63] in Ctrl samples (black), sALS samples with regular STMN2 levels (STMN2 expression Z-score > −2, purple), and sALS samples with reduced STMN2 levels (STMN2 expression Z-score < −2, red). Black horizontal lines illustrate median and interquartile ranges. Statistical significance was determined by Kruskal-Wallis test and Dunn's post hoc test to identify changes between individual groups. *P value < 0.05; **P value < 0.01; ***P value < 0.001; ****P value < 0.0001

Fig 3: Binding of NOVA1, NOVA2 and RBFOX2 is associated with AS events that are differentially regulated in ALS. a Schematic illustrating the experimental and analysis workflow n = 2 Ctrl per eCLIP). b Pie charts depicting distribution of gene regions bound by eCLIP-seq peaks in a dataset. The total number of significant peaks is shown below the individual pie charts. c Heatmaps illustrating the percentage of individual peaks (top) and target transcripts (bottom) shared between the indicated eCLIP-seq datasets. d–g Scatter plot presenting k-mer enrichments as Z-scores of d TDP-43 (6-mers), e NOVA1 (4-mers), f NOVA2 (4-mers), and g RBFOX2 (6-mers) in the Ctrl-1-2 (x axis) and the CV-B (y axis) dataset, respectively. k-mers with highest enrichment are colored. h–l Bar graphs illustrating enrichment significance (left graph, −log10(P value))) and fold-enrichment (right graph) of TDP-43 (green), NOVA1 (red), NOVA2 (orange) and RBFOX2 (blue) eCLIP-seq experiments in CV-B (dark shading) and Ctrl-1-2 (light shading) at AS events called in the h sALS vs. Ctrl dataset generated in this study, i fALS vs. Ctrl dataset generated in this study, j sALS vs. Ctrl dataset from AnswerALS, k C9-ALS vs. Ctrl dataset from AnswerALS, and l SPG11-HSP vs Ctrl generated in this study. Dashed vertical lines indicate significance thresholds (P value = 0.05 and fold enrichment=1). Hypergeometric test was used for calculation of significance. All detected events that passed the coverage threshold in the RNA-seq datasets were used as background

Fig 4: NOVA1 expression is increased in ALS iPSC-MN and elevated cytoplasmic TDP-43 levels are associated with a reduction of NOVA1 mRNA levels. a–d Bar graphs of gene expression values for NOVA1 as reads per kilobase transcript per million mapped reads (RPKM) from a sALS and Ctrl generated in this study (n = 4 sALS, n = 4 Ctrl), b fALS and Ctrl generated in this study (n = 2 fALS, n = 3 Ctrl), c sALS and Ctrl from the AnswerALS consortium dataset (n = 9 sALS, n = 9 Ctrl), and d C9-ALS and Ctrl from the AnswerALS consortium dataset (n = 5 C9-ALS, n = 9 Ctrl). Data presented as mean ±SD. Statistical significance was determined by Mann-Whitney U test. a P value = 0.0286, b P value = 0.2000, c P value = 0.0315, d P value = 0.0599. e Western blot from total cell lysates of iPSC-MNs stained for NOVA1 and GAPDH. f Bar graph showing densitometric quantification of Western blot of NOVA1 normalized to GAPDH (n = 6 ALS; n = 6 Ctrl). Fold change ALS/Ctrl: 1.42. Data presented as mean ±SD. Statistical significance was determined by Mann-Whitney U test. P value = 0.0152. g Bar plot with NOVA1 expression values (in TPM) in iPSC-MN with control siRNA or siRNA targeting TARDBP (GEO ID: GSE121569; n = 6 control siRNA, n = 6 TARDBP siRNA). Data presented as median +IQR. Statistical significance was determined by Mann-Whitney U test. P value = 0.3095. h Bar plot with NOVA1 expression values (in TPM) in SH-SY5Y with control siRNA or siRNA targeting TARDBP (GEO ID: GSE122069; n = 3 control siRNA, n = 3 TARDBP siRNA). Data presented as median +IQR. Statistical significance was determined by Mann-Whitney U test. P value = 0.4000. i Bar plot with NOVA1 expression values (in RPKM) in mouse striatum with control ASO or ASO targeting TARDBP (GEO ID: GSE27394; n = 4 control ASO, n = 5 TARDBP ASO). Data presented as median +IQR. Statistical significance was determined by Mann-Whitney U test. P value = 0.9048. j Bar plot with NOVA1 expression values (in RPM) in mouse brain without and with TDP-43-dNLS expression (GEO ID: GSE65973; n = 4 Ctrl, n = 4 TDP-43-dNLS). Data presented as median +IQR. Statistical significance was determined by Mann-Whitney U test. P value = 0.0286. k Bar plot with NOVA1 expression values (in DESeq2 normalized counts) in iPSC-MN (4 Ctrl, 4 HNRNPA2B1 mutant lines) without and with puromycin stress (GEO ID: GSE157467; n = 8 untreated, n = 8 puromycin). Data presented as median +IQR. Statistical significance was determined by Mann-Whitney U test. P value = 0.0379

Fig 5: NOVA1 mediates AS in human iPSC motor neurons in a position-dependent manner. a Schematic illustrating the experimental workflow. b Western blot of NOVA1 from day 30 iPSC-MN after transduction with lentivirus containing EGFP (n = 3) or NOVA1 (n = 3) ORF expressed under the CAG promoter. GAPDH was used as a loading control. Average fold change of 1.97 (CV-B: 2.2-fold; Ctrl-1-1: 1.6-fold; Ctrl-2-1: 2.1-fold). c Western blot of NOVA1 from day 30 control (n = 5) and NOVA1 K.O. (n = 5) iPSC-MN. GAPDH was used as a loading control. d Venn diagram of significant differential cassette exon alternative splicing events from comparisons of NOVA1 vs. EGFP overexpression (blue) and of NOVA1 wt vs. NOVA1 K.O. (yellow). Significance of overlap was calculated with Fisher's exact test using all events detected in both analyses as background. OR = odds ratio. e, f Bar graphs of significance of differential enrichment of eCLIP-seq binding sites at AS events of TDP-43 (green), NOVA1 (red), NOVA2 (orange) and RBFOX2 (blue) in e NOVA1 vs EGFP overexpression, and f NOVA1 K.O. vs Ctrl. Experiments were performed in two cell lines (CV-B, dark shading; Ctrl-1-2, light shading). Dashed horizontal line at P value = 0.05. Enrichment was calculated using hypergeometric test with all detected events as the background. g Schematic illustrating analysis of positions of interest for NOVA1 binding analysis. h-i Bar graphs of significance of differential enrichment of NOVA1 eCLIP-seq binding sites at excluded (left) and included (right) AS events (upstream exons, upstream introns, alternative exons, downstream introns and downstream exons) in h NOVA1 vs EGFP overexpression, and i NOVA1 K.O. vs Ctrl. Experiments were performed in two cell lines (CV-B, dark shading; Ctrl-1-2, light shading). Dashed vertical line at P value =0.05. Enrichment was calculated using hypergeometric test with all detected events as the background. j Model illustrating physiological modes of action of NOVA1 in human iPSC-derived MN. AE alternative exon, UI upstream intron, DI downstream intron