Fig 2: Extensive AS events occur in response to Strap loss in lineage-committed EB cells.a Dot plot showing distribution of ?PSI values (?PSI = PSI(Strap-/-)–PSI(WT)) for each splicing category. The value of ?PSI between WT and Strap-/- EBs was based on two independent biological samples from each group. b Splicing heatmap showing the values of PSI for exon-skipping events between WT (n = 2, biological replicates) and Strap-/- (n = 2, biological replicates) EBs. c GO enrichment analysis showing top ranked biological functions for alternatively spliced genes between WT and Strap-/- EBs (9-day-old). d A schematic of the alternative and constitutive exons. 5' and 3' splicing sites (5',3' ss) are indicated accordingly. e Box plots of splice site scores calculated for STRAP-regulated skipped exons. Boxplots show median (the horizontal line in the box), 25 and 75% percentiles (lower and upper bounds of box, respectively), minimum and maximum (lower and upper whiskers, respectively). P-values were determined by two-sided unpaired Wilcoxon tests. For ALT3’ss, unresponsive n = 79, enhanced n = 61, repressed n = 53; for ALT5’ss, unresponsive n = 67, enhanced n = 53, repressed n = 33; for CON1 5’ss, unresponsive n = 67, enhanced n = 73, repressed n = 53; for CON2 3’ss, unresponsive n = 73, enhanced n = 82, repressed n = 63. f Hierarchically clustering matrix showing the correlation for STRAP and other known splicing factors. Spearman correlation coefficients were calculated based on the ?PSI values of skipping exons affected by either STRAP or previously reported splicing factors36. Highly correlated factors are highlighted in red. g Validation by RT-PCR of genes with SE events identified by MAJIQ. Information for target exons is shown on the right panel. Empty box, constitutive exon; black and red boxes, skipped exon. Data are pooled from two independent experiments (n = 4 per group) and error bars represent the mean ± SD (right panel). Unpaired two-tailed Student’s t-test was used. h Venn diagram showing the intersection of AS events between rMATS and MAJIQ datasets. The detailed AS category and respective numbers are shown at the bottom. P-values were determined by Chi-squared test.

Fig 3: Intracellular STRAP binds with subunits of the spliceosome complex.a List of STRAP-binding proteins for spliceosome complex subunits. Anti-STRAP antibody was used to immunoprecipitate STRAP and co-precipitate its interacting proteins from mouse ESC nuclear extracts (NE). The binding partners were identified by LC-MS/MS. b Ranked curve for STRAP binding-partners (see method). Enriched complexes (colored dots) are those that meet the P-value cutoff (P < 5.012e-2). Top enriched complexes are indicated. c Interactions between indicated proteins and STRAP were confirmed by co-immunoprecipitation and Western blotting analyses. Samples were prepared as in (a) and treated with or without ribouclease A (RNase A). 1% of lysates were loaded as input control. The levels of co-precipitated proteins after RNase treatment (relative to untreated control) are shown below the gel pictures. The experiment was independently repeated twice with similar results. d The localization of STRAP (green) and indicated proteins (red) in mouse ESC cells were analyzed by immunofluorescence. Cell nuclei were labeled by DAPI (blue) staining. Scale bar, 10 µm. Images were captured by a Keyence microscope. The experiment was repeated three times independently with similar results.

Fig 4: Loss of STRAP has negative effects on 17S U2 snRNP biogenesis.a, b Representative Western blots (a) and protein-level quantifications (b) of several spliceosomal components in WT and STRAP KO cells at the indicated days. All data were normalized to ß-Actin. Data are shown from one of two independent experiments; bars represent as means (n = 2, biological replicates). c–f Representative Western blots (c and e) and co-immunoprecipitated protein-level quantifications (d and f) showing co-IP results. All data were normalized to the respective input. The expression levels of indicated proteins (relative to its parallel WT controls) are shown below the gel pictures. Data, pooled from two independent experiments; bars represent as means. g Schematic illustration of STRAP-truncated mutants (upper panel) and co-IP experiments to map the domain(s) of STRAP required for binding of SR140 (bottom panel). 293T cells were transfected either with empty vectors or expression vectors as indicated. Cellular extracts were then subjected to immunoprecipitation with an HA antibody followed by Western blot analysis using a Flag or HA antibody. Expressions of transfected proteins were also determined. The experiment was repeated three independent times with similar results. h Gain-of-function assay using UPF3A mini-gene in MEF WT and Strap KO cells. Indicated cells were co-transfected with miniUPF3A and an empty vector or a vector encoding various Flag-tagged STRAP, as shown in g. RT-PCR assays were performed to detect alternative exon inclusion in miniUPF3A. Quantifications of PSI values are shown below the gels, and the fractions of mis-splicing are pooled from three independent analyses (n = 3). Error bars represent the mean ± SD. The Flag-tagged protein levels and ß-Actin were assayed by Western blot analysis. ß-Actin was used as a loading control. P-values were determined by unpaired two-tailed t-tests.

Fig 5: STRAP-RNA interaction map showing direct binding to skipped exons.a Venn diagram showing the intersection of STRAP-regulated AS genes (defined by rMATS and MAJIQ) and genes within STRAP eCLIP peaks. b, c Composite maps for STRAP-binding peaks on AS events based on the RNA-seq data (|?PSI|> 0.1, FDR < 0.05). d At day 12, 40% of WT EBs showed an immature action potential (AP) in response to depolarizing current steps and a prominent sag in response to hyperpolarizing current step (red traces). At day 22, 85% of cells showed AP, potential sag, and “rebound” APs (dashed box in middle red traces). Some cells showed spontaneous AP firing at RMP (bottom red traces). e 20% of KO EBs showed immature AP at day 12 and 25% at day 22. f Venn diagram showing the intersection of STRAP-regulated SE genes defined by rMATS (FDR < 0.1) and genes within STRAP eCLIP peaks. g IGV viewer genome browser image of eCLIP signals on Nnat pre-mRNA. h RIP assay for STRAP binding on Nnat in EBs. Targeted regions (black outlined box) with respective paired primers are shown at the top. i, j RT-PCR quantified PSI values of Nnat in EBs. k IGV viewer genome browser image of eCLIP signals on Mark3 pre-mRNA. l RIP assay for STRAP binding on Mark3 pre-mRNA. m, n RT-PCR quantified PSI values of Mark3 in EBs. o RT-PCR quantified PSI values of Nnat and Mark3 in E14 cell line-derived EBs at day 14. Left bottom, Western blot analysis showing STRAP expression in inducible STRAP knockdown EBs with ß-Actin as a loading control. For (i, m), and (o), PSI values are shown below gel pictures. Experiments were repeated three independent times with similar results. For (h) and (l), error bars show the mean ± SD from n = 3 technical replicates. Experiments were repeated twice with similar results. For (j) and (n), data are pooled from two independent experiments (n = 4) and error bars represent the mean ± SD. For (o), data are pooled from three independent experiments (n = 3) and error bars represent the mean ± SD. P-values were determined by unpaired two-tailed t-tests.