Figure 1. RBPMS is associated with SMC super-enhancers and is highly expressed in the differentiated PAC1 cells.

(A) Diagram of the SMC dedifferentiation. SMC markers of differentiation and dedifferentiation are respectively shown at the bottom and top of the diagram. (B) Venn diagram of RBP genes associated with super-enhancers across different human smooth muscle tissues. Skeletal muscle was used as an outlier. RBPs common to all smooth muscle tissues but not skeletal muscle are shown on the left. (C) Schematic of the AS event determining the two major RBPMS isoforms, RBPMS A (red) and RBPMS B (yellow). (D) RT-PCR analysis of SMC splicing markers, Actn1 and Tpm1, in differentiated (D) and proliferative (P) PAC1 cells. Schematic of the regulated mutually exclusive splicing events on top and respective isoforms products on the left. Values shown are the quantified PSI (percent spliced in) of the smooth muscle isoforms (SM) ± standard deviation (n = 3). (E) qRT-PCR analysis of Rbpms (all isoforms), Rbpms2 and SMC differentiation markers Acta2, Cnn1 and Smtn, in PAC1 cells D (green) and P (blue). 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. Statistical significance was performed using Student’s t-test (*p<0.05, **p<0.01, ***p<0.001). (F) Western blots for RBPMS in D and P PAC1 cells. ACTA2 is a SMC differentiation marker and GAPDH a loading control. A and B indicates the two RBPMS isoforms. (G) Immunofluorescence in D and P PAC1 cells for RBPMS. DAPI staining for nuclei.

Figure 1—figure supplement 1. RBPMS is highly expressed in SM tissues.

(A) RBPMS mRNA expression across different human tissues in the GTEX database (GTEx Consortium, 2013). TPM: transcripts per million.

Figure 1—figure supplement 2. mRNA abundance analysis of the rat aorta SMC de-differentiation RNA-Seq.

(A) Schematic of the experimental design of the rat aorta tissue dedifferentiation. (B) mRNA abundance of RBP genes in differentiated aorta tissue (T) and proliferative passage 9 (P9). RBPs whose genes were found associated with smooth muscle tissue super-enhancers (Figure 1B) are highlighted in green. Additionally, other well characterized RBPs and Rbpms2 are labelled in black. LOC108348175 is the Ensembl annotated rat Qki like gene. Statistical significance in mRNA abundance changes between T and P9 was calculated by DESeq2 and is indicated in dark and light gray color (padj <0.05 and non-significant changes respectively). mRNA levels are expressed in transcripts per million (TPM). Cpsf4l gene was not found in this dataset. (C) mRNA abundance changes measured in transcripts per million (TPM) during rat aorta dedifferentiation (T, SC, P0 and P9). Genes shown are Rbpms and its paralog Rbpms2, SMC differentiation markers, Acta2, Cnn1 and Smtn, and other RBPs, Mbnl1, Ptbp1 and Qk1.

Figure 1—figure supplement 3. Alternative splicing analysis of the rat aorta SMC de-differentiation RNA-Seq.

(A) AS changes when comparing T and P9 conditions. Only significant ASE with FDR < 0.05 and ΔPSI greater than 10% are shown. ASE were classified as skipped exons (SE), mutually exclusive exons (MXE), 5’and 3’ alternative splice sites (A5SS and A3SS) and retained intron (RI). The number of events differentially alternatively spliced are shown in the graph. (B) Overlap of the number of genes affected at the levels of mRNA abundance and AS in the T and P9 comparison. (C) Sashimi plot of the SMC mutually exclusive splicing markers, Actn1 and Tpm1, in rat aorta tissue dedifferentiation. PSI values represent the mean of the PSI values calculated by rMATS. (D) RBPMS motif enrichment in differentially alternatively spliced SE in the T and P9 comparison. A pair of CAC separated by 1 to 12 nt was used as the RBPMS motif. Values indicate the motif enrichment. Statistical significance was calculated by the Matt tool (*p<0.05, **p<0.01, ***p<0.001).