Fig. 6
SRSF3 promotes dPAS usage by maintaining high levels of CFIm. a Volcano plot of differential gene expression after Srsf3 KD analyzed by DESeq2. Significant genes (adjusted P value ≤ 0.1, log2-transformed fold change ≥ 0.5) are highlighted in blue. Genes of CPA factors, Srsf3 and Srsf7, are indicated with significant changes highlighted in bold. b Quantification of Cpsf6 mRNA levels after KD of Srsf3 and Srsf7 by RT-qPCR. Data are represented as mean ± stDev. n = 3, Student’s t test. ***P value < 0.005, **P value < 0.01. c Top: Sashimi plots and coverage of RNA-seq reads mapping to Cpsf6 exons 5 to 7 in control samples (WT, gray) and after KD of Srsf3 (blue) and Srsf7 (orange). Bottom: Genome browser view of iCLIP crosslink events of SRSF3 from P19 cells in the same region. Small alternative exon within intron 5 (termed ‘exon 5a’, 111 nt) indicated in light blue. d RT-PCR after KD of Srsf3 and Srsf7 using primers in exons 5 and 7 of Cpsf6. Cpsf6 isoforms corresponding to the PCR amplicons are indicated beside gel. Shorter exon 6-skipped isoforms are indicated with asterisks. CalR amplicons served as control for cDNA integrity. e Western blot using P19 cell lysates after KD of Srsf3 and Srsf7. Protein levels of CPSF5, CPSF6, SRSF3, and SRSF7 were assayed using specific antibodies. Alpha-tubulin was used as loading control. f Quantification of CPSF6, CPSF5, and SRSF3 protein levels from Western blots. n = 3. Student’s t test. ***P value < 0.001, *P value < 0.05. g Scatterplot of PDUIs in P19 control cells (Ctrl) and after Cpsf6 KD. Genes with significant changes in 3′UTR-APA (adjusted P value ≤ 0.1) are highlighted in green. h Overlap of genes with shortened 3′UTRs after KD of Srsf3 or Cpsf6. i Scatter plot comparing dPAS usage of KD Srsf3 and Cpsf6