PDGFRα signaling for one hour minimally affects gene expression.

(A) Schematic of RNA-seq experimental design. iMEPM cells were transduced to stably express a scramble shRNA (scramble) or shRNA targeting the 3’ UTR of Srsf3 (shSrsf3). iMEPM cells expressing either scramble or shSrsf3 were left unstimulated or stimulated with 10 ng/mL PDGF-AA for 1 hour and RNA was isolated for RNA-seq analysis. (B) Western blot (WB) analysis of whole-cell lysates from scramble and shSrsf3 cell lines with anti-Srsf3 and anti-Gapdh antibodies. The percentage of Srsf3 expression normalized to Gapdh expression is indicated below. (C) Volcano plots depicting differentially-expressed genes in scramble versus shSrsf3 cell lines in the absence (left) or presence (right) of PDGF-AA stimulation. Log2(fold change) (FC) values represent log2(shSrsf3 normalized counts/scramble normalized counts). Significant changes in gene-level expression are reported for genes with adjusted P (padj) < 0.05 and fold change |FC| ≥ 2. (D) Venn diagram of significant genes from C. (E) Volcano plots depicting differentially-expressed genes in the absence versus presence of PDGF-AA ligand in scramble (left) or shSrsf3 (right) cell lines. Log2(FC) values represent log2(+PDGF-AA normalized counts/-PDGF-AA normalized counts). (F) Venn diagram of significant genes from E.

PDGFRα signaling for one hour has a more pronounced effect on alternative RNA splicing.

(A) Volcano plots depicting alternatively-spliced transcripts in scramble versus shSrsf3 cell lines in the absence (left) or presence (right) of PDGF-AA stimulation. Difference in percent spliced in (ΔPSI) values represent scramble PSI – shSrsf3 PSI. Significant changes in alternative RNA splicing are reported for events with a false discovery rate (FDR) ≤ 0.05 and a difference in percent spliced in (|ΔPSI|) ≥ 0.05. (B) Venn diagram of significant transcripts from A, filtered to include events detected in at least 10 reads in either condition. (C) Volcano plots depicting alternatively-spliced transcripts in the absence versus presence of PDGF-AA ligand in scramble (left) or shSrsf3 (right) cell lines. Difference in percent spliced in (ΔPSI) values represent -PDGF-AA PSI – +PDGF-AA PSI. (D) Venn diagram of significant transcripts from C, filtered to include events detected in at least 10 reads in either condition. (E) Bar graph depicting alternative RNA splicing events in scramble versus shSrsf3 cell lines in the absence or presence of PDGF-AA stimulation (left) or in the absence versus presence of PDGF-AA ligand in scramble or shSrsf3 cell lines (right).

Srsf3 exhibits differential transcript binding upon PDGFRα signaling.

(A) Schematic of eCLIP experimental design. iMEPM cells were left unstimulated or stimulated with 10 ng/mL PDGF-AA for 1 hour and processed for eCLIP analysis. (B) Immunoprecipitation (IP) of Srsf3 from cells that were UV-crosslinked or not UV-crosslinked with IgG or an anti-Srsf3 antibody followed by western blotting (WB) of input, supernatant (Sup), and IP samples with an anti-Srsf3 antibody. (C) Mapping of eCLIP peaks to various transcript locations in the absence or presence of PDGF-AA stimulation. 5’ UTR, 5’ untranslated region; CDS, coding sequence; 3’ UTR, 3’ untranslated region. (D,E) Mean coverage of eCLIP peaks across various transcript locations (D) and surrounding the 5’ and 3’ splice sites (E) in the absence or presence of PDGF-AA stimulation. (F,G) Top three motifs enriched in eCLIP peaks in the absence (F) or presence (G) of PDGF-AA stimulation.

Srsf3 and PDGFRα signaling are associated with differential GC content and length of alternatively-spliced exons.

(A) Box and whisker plot depicting the percentage of exon GC content in exons that are not differentially alternatively spliced, and exons that are included or skipped when Srsf3 is present from the rMATS analysis. (B) Box and whisker plot depicting the ratio of downstream intron to exon GC content in exons that are not differentially alternatively spliced, and exons that are included or skipped when Srsf3 is present from the rMATS analysis. (C,D) Box and whisker plots depicting the ratio of upstream intron to exon length (C) and downstream intron to exon length (D) in exons that are not differentially alternatively spliced, and exons that are included or skipped when Srsf3 is present from the rMATS analysis. (E) Violin and box and whisker (inset) plots depicting the percentage of exon GC content in exons that are not bound by Srsf3, and exons that are bound in the absence and/or presence of PDGF-AA stimulation from the eCLIP analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Transcripts bound by Srsf3 that undergo alternative splicing upon PDGFRα signaling encode regulators of PI3K signaling.

(A) Venn diagram of genes with differential expression (DE) or transcripts subject to alternative RNA splicing (AS) across the four treatment comparisons that overlap with transcripts with Srsf3 eCLIP peaks in the absence or presence of PDGF-AA stimulation. (B,C) Top ten (B) and PI3K-related (C) biological process gene ontology (GO) terms for transcripts from the overlapping datasets. p.val, P. (D) Difference in percent spliced in (ι1PSI) values for PI3K/endosome-related transcripts of interest. FDR, false detection rate. (E) Peak visualization for input and eCLIP samples in the absence or presence of PDGF-AA stimulation from Integrative Genomics Viewer (left) with location of motifs from Figure S5 indicated below for PI3K/endosome-related transcripts of interest. Predicted alternative splicing outcomes for PI3K/endosome-related transcripts of interest (right).

Srsf3 regulates early endosome size and phosphorylation of Akt downstream of PDGFRα signaling.

(A,B) Scatter dot plots depicting average size of Rab5 puncta per cell (A) and Pearson’s correlation coefficient of signals from anti-Rab5 and anti-PDGFRα antibodies (B) in scramble and shSrsf3 cell lines in the absence or presence (15-60 min) of PDGF-AA stimulation. Data are mean ± s.e.m. *, P < 0.05. Shaded shapes correspond to independent experiments. Summary statistics from biological replicates consisting of independent experiments (large shapes) are superimposed on top of data from all cells; n = 20 technical replicates across each of three biological replicates. (C-H”) PDGFRα antibody signal (white or magenta) and Rab5 antibody signal (white or green) as assessed by immunofluorescence analysis of scramble and shSrsf3 cells in the absence or presence (15-60 min) of PDGF-AA stimulation. Nuclei were stained with DAPI (blue). White arrows denote colocalization. Scale bars: 20 μm (main images), 3 μm (insets). (I) Western blot (WB) analysis of whole-cell lysates (WCL) from scramble (left) and shSrsf3 (right) cell lines following a time course of PDGF-AA stimulation from 15 min to 4 h, with anti-phospho-(p)-Akt and anti-Akt antibodies. Line graphs depicting quantification of band intensities from n = 3 biological replicates as above. Data are mean ± s.e.m. *, P < 0.05; **, P < 0.01. (J) Model of experimental results in which PI3K/Akt-mediated PDGFRα signaling results in the nuclear translocation of Srsf3 and the subsequent AS of transcripts to decrease levels of proteins that promote PDGFRα trafficking out of early endosomes.