Figure 5. RBPMS regulates functionally important targets in SMCs.

(A) GO analysis of genes with cassette exons regulated in RBPMS knockdown. The top five enriched GO terms in the three categories (biological process, molecular function and cellular component) are shown. Values within and in front of the bars indicate the number of genes in the enriched term and the enrichment relative to the background list. (B) Enrichment of exons regulated by RBPMS knockdown within genes associated with super-enhancers in smooth muscle tissues. Background set is all cassette exon events (regulated and unregulated) detected by rMATS in the same experiment. Significance determined by hypergeometic P-value. (C) PPI network of genes showing concordant splicing regulation upon RBPMS knockdown and PAC1 differentiation status, combined with genes concordantly regulated by RBPMS overexpression and in aorta tissue datasets. PPI network was generated in STRING using experiments and database as the sources of interactions. Network edges represent the interaction confidence. Enriched GO terms (BP, biological process, MF, molecular function and CC, cellular component) were also included in the analysis and are indicated in red, blue and yellow. Super-enhancer associated gene names are in bold and are highlighted gray, light green or dark green shading according to whether they were super-enhancer associated in 1, 2 or 3 SMC tissues. (D) Immunofluorescence of RBPMS and actin (Phalloidin) in differentiated and proliferative PAC1 cells (D and P) and upon prolonged (120 hr) RBPMS knockdown in differentiated PAC1 cells (D Ctr and D KD). DAPI staining for cell nuclei. Scale bars 10 μm. (E) Left, anisotropy measurement of actin fibers in PAC1 cells D (differentiated) and P (proliferative) using the FibrilTool ImageJ macro (n = 44 and 52). Middle, nucleus size measurement shown in pixels (n = 182 and 130). Right, average of the cell size quantified per field (n = 11 and 15). (F) Left, anisotropy measurement of actin fibers in RBPMS knockdown (D Ctr and D KD) using the FibrilTool ImageJ macro (n = 56 and 33). Middle, nucleus size measurement shown in pixels (n = 363 and 75). Right, average of the cell size quantified per field (n = 14 and 10). In (E) and (F), statistical significance was obtained from a Mann-Whitney-Wilcoxon Test (*p<0.05, **p<0.01, ***p<0.001). Data shown are from one representative experiment carried out in triplicate.

Figure 5—figure supplement 1. GO analysis of genes differentially spliced upon RBPMS overexpression and PAC1 and aorta dedifferentiation.

Top five enriched GO terms from Gorilla GO analysis of differentially spliced genes. (A) RBPMS overexpression in proliferative PAC1 cells. (B) PAC1 dedifferentiation. (C) Rat aorta tissue dedifferentiation. Values within and in front of the bars indicate the number of genes in the enriched term and the enrichment relative to the background list.

Figure 5—figure supplement 2. Morphological changes in PAC1 cells are specific to RBPMS knockdown.

(A) Immunofluorescence of RBPMS and ACTIN (Phalloidin) in PAC1 cells treated with a second siRNA only (KD3, Stealth siRNAs, Thermo Fisher Scientific (RSS363830): CAG UAC UCC UCU GCC CAA CAC UGU A) and combined with siRNA1 (KD1 and 3–45 pmols of each) after 120 hr siRNA treatment. DAPI staining for cell nuclei. Scale bars 10 μm. (B) Left, anisotropy measurement of ACTIN fibers in RBPMS knockdown (C, K1, K3 and K1 and 3) using the FibrilTool ImageJ macro (n = 56, 33, 26, 33, respectively). Middle, nucleus size measurement shown in pixels (n = 363, 75, 82, 93, respectively). Right, average of the cell size quantified per field (n = 14, 10, 7, 10). Statistical significance was obtained from a Mann-Whitney-Wilcoxon Test (*p<0.05, **p<0.01, ***p<0.001). Data shown are from one representative experiment carried out in triplicate.

Figure 5—figure supplement 3. Sustained RBPMS knockdown in PAC1 cells.

(A) Western blots showing RBPMS knockdown in PAC1 cells after 120 hr siRNA treatment (control (C), siRNA1 (KD1), siRNA3 (KD3), siRNA1 and 3 (KD1 and 3)). Antibody targeting ACTA2, a SMC differentiation marker, and GAPDH was used as loading control. (B) qRT-PCR analysis of Rbpms (all isoforms), and the SMC differentiation marker Acta2, in knockdown (control (C), siRNA1 (KD1), siRNA3 (KD3), siRNA1 and 3 (KD1 and 3)). Relative expression was normalized to the average of two housekeepers (Gapdh and Rpl32) and the mean of the relative expression is shown (n = 3). Each point shows data from an individual sample. Changes in Acta2 were not significant. (C) RT-PCR analysis of RBPMS splicing targets Actn1 and Ptprf, in PAC1 cells after 120 hr RBPMS KD. Schematic of the regulated isoform products on the left. Values shown are the quantified PSI (percent spliced in) of the smooth muscle isoforms (SM). Data are from one representative experiment performed in triplicate. Statistical significance was assessed using Student’s t-test (*p<0.05, **p<0.01, ***p<0.001).