Fig 2: Comparison of SF3B1 and SF1 modulation of representative U2AF2-responsive gene transcripts. The effects of siRNA-mediated reductions in SF1, SF3B1, or U2AF2 levels on splicing of representative U2AF2-regulated sites, including A, THYN1; B, SAT1; C, INTS13; and D, RNF10, were compared by RT-PCR as described for Figure 6. n.s., not significant; ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005.

Fig 3: Similarity of Splicing Factor 1 (SF1) and Quaking (QKI) and RNA-seq analysis of SF1 or QKI loss-of-function in HepG2 cells.(A) Overlay of SF1 (cyan) and QKI (tan) KH and QUA2 protein domains: green highlighted region indicates KH and QUA2 domains of QKI; the two tan alpha helices on the left side make up the QUA1 domain in QKI (which SF1 lacks; text indicates residues in unstructured regions of QKI). (B) Vast-tools analysis of the ENCODE RNA-seq data from SF1 (shSF1) or QKI shRNA (shQKI) knockdown compared to control shRNA (shNT) in HepG2 cells; the y-axis shows dPSI for shQKI or shSF1 relative to shNT for significantly altered alternatively spliced exons (AltEX; left) or intron retention (IR; right; dPSI > |10| and MVdPSI > 0), and ****p<0.0001 by Mann-Whitney U when comparing the distribution of changes in shQKI relative to shNT to shSF1 relative to shNT. (C) Scatter plot showing the distribution of AltEX events that changed under depletion of both SF1 (dPSI values relative to shNT on the y-axis) and QKI (dPSI values relative to shNT on the x-axis); the number in each quadrant indicates how many AltEX events were observed; RAI14 exon 11 is shown in cyan. (D) Simple Enrichment Analysis (SEA) of the intron region spanning 60 nt to 20 nt upstream of the 3’ss in QKI and SF1 regulated AltEX events shown in C (p<0.05). (E) rMAPS motif map for UACUAA generated for AltEX events changing during shSF1 compared to shNT by rMATS; motif scores (solid line) or -log10 p-value (dotted line) is shown in red for exons whose inclusion increases and in blue for exons whose inclusion decreases. (F) rMAPS motif map as described in E. but for shQKI treatment compared to shNT.

Fig 4: Analysis of the proteins associating with RAI14 intron 10 RNA.RNA affinity chromatography (RAC) with liquid chromatography and tandem mass spec or western blot analysis. (A) Schematic representation of substrates used for RNA affinity chromatography (RAC) which included 64 nt of RAI14 intron 10, 6 nt of exon sequence, and the tobramycin aptamer (tobra): wild-type, upDEL, dnDEL, 2xDEL, and aptamer only; C2C12 nuclear extract (NE) was incubated with these, and RAC-associated eluates were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) or western blot. (B) Heatmap showing hierarchical clustering of early spliceosome and 17S U2 snRNP protein (E/U2) abundance detected in the RAC-LC-MS/MS datasets for each substate shown and in the presence of ATP; these represent background-corrected levels relative to NE (see Methods) and the scale bar shows row Z-score values. (C) Numbers observed for relative levels of E/U2 proteins (background corrected relative to NE) observed associating with each RAC substate with either a positive (npos) or negative (nneg) value. (D) Western blot of NE (input) or WT, dnDEL (dn), or 2xDEL (2 x) RAC time-course (+ATP as described in B and C) for 7.5 min (left), 15 min (middle), or 30 min (right) probed with anti-panQki (top) or anti-SF1 (bottom) antibodies; MW shown in kD to the right. (E) Heatmap showing hierarchical clustering of early spliceosome and 17S U2 snRNP protein (E/U2) abundance detected in the RAC-LC-MS/MS datasets for each substate shown and in the absence of ATP; these represent data-independent acquisition (DIA; see methods) values normalized to NE and each mutant is shown as log2 fold change relative to the WT substate and passed cutoff of log2 fold change > |0.2| and p<0.01. (F) Volcano plot comparing LC-MS/MS log2 protein abundance (log2 fold-change (log2FC); x-axis) of E/U2 (circles) and other RNA-binding proteins (RBPs) (squares) observed associating with RAC substates dnDEL compared to WT (y-axis shows -log10 p-value) of enriched proteins (cutoff: L2FC > |0.7| and p<0.01); inset shows the number observed for those enriched in dnDEL (yellow) or WT (blue); schematic below shows model of RAC substates recruitment to distinct protein-associated species. (G) Western blot of NE (input) or WT, dnDEL (dn), or 2xDEL (2 x) RAC time-course (-ATP as described in E and F) for 7.5 min (left), 15 min (middle), or 30 min (right) probed with anti-panQki (top), anti-SF1 (middle), or anti-TATSF1 (bottom); MW shown in kD to the right. Figure 4—source data 1.Original membranes corresponding to western blots depicted in Figure 4D (left), or Figure 4G. Figure 4—source data 2.Original membranes corresponding to western blots depicted in Figure 4D (left), or Figure 4G.

Fig 5: RAI14 exon 11 is repressed by QKI and activated by Splicing Factor 1 (SF1).(A) UCSC Genome Browser screenshot (top panel) with RNA-seq reads mapping to RAI14 for shQKI (top), shNT (middle), or shSF1 (bottom); percent spliced in (PSI) values are measured by Vast-tools; the inset shows boxed regions of (from top) two ACUAAC elements with Cola-seq branchpoints mapping to one nucleotide downstream of a branchpoint adenine, ACUAA elements by oligomatch, then Quaking (QKI) eCLIP peaks from K562 (top) or HepG2 (bottom) cells that overlap with these; conservation of 100 vertebrates is shown at the bottom. (B) Western blot of protein extracted from HEK293 QKI KO cells (top) probed with anti-tubulin (magenta) or anti-panQKI (green) antibodies (MW in kD is shown on right); RT-PCR of RAI14 exon 11 and BioAnalyzer gel-like image (bottom) of RNA extracted from the cells above showing mean percent included ± standard deviation below (n=3 biological replicates; ****p<0.001 by Student’s t-test compared to ctrl; size of amplicon in bp is shown on right). (C) Western blot (top) of proteins extracted from HEK293 cells transfected with siNT, siSF1_2, or siSF1_1+2, probed with anti-SF1 (green) or anti-Gapdh (magenta) antibodies; the protein abundance (fold change relative to the siNT control ± standard deviation) is shown below and MW in kD is shown on right; RT-PCR of RAI14 exon 11 and BioAnalyzer gel-like image (bottom) of RNA extracted from the cells described above with mean percent included ± standard deviation below (n=3 biological replicates; **p<0.01 or ***p<0.001 by Student’s t-test compared to siNT; size of amplicon in bp is shown on right). (D) Western blot (top) of proteins extracted from C2C12 myoblasts transfected with siNT, siQki or siSf1_1+2, probed with anti-SF1 (magenta; top), anti-tubulin (magenta; middle) or anti-panQki (green; bottom); MW in kD is shown on right. The protein abundance (fold change relative to the siNT control ± standard deviation) is shown below (n=3 biological replicates; **p<0.01 or ***p<0.001 by Student’s t-test); RT-PCR of Rai14 exon 11 and BioAnalyzer gel-like image (bottom) of RNA extracted from the C2C12 cells described above with mean percent included ± standard deviation indicated below (****p<0.0001 by Student’s t-test; size of amplicon in bp is shown on right). Figure 2—source data 1.Original membranes and BioAnalyzer gel-like images corresponding to Figure 2A (left), Figure 2C (middle), or Figure 2D (right).See text under each set of images for additional details. Figure 2—source data 2.Source data containing original uncropped western blots and BioAnalyzer gel-like images/files.