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. 2022 Jul 1;144(3):413–435. doi: 10.1007/s00401-022-02450-3

Fig. 4.

Fig. 4

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