Fig 2: Event-based splicing analysis highlights potential RBPMS-dependent events downstream of MYOCD. The output from our rMATS analysis was overlapped with genes found to undergo Rbpms-dependent splicing in rat cells in a previous study [35]. A Genes from the overlap of these datasets. For confirmation of true splicing events, we used event-specific primers and PCR. Amplicons were separated on agarose gels (B, n = 5–6). Events are ordered by absolute change in percent spliced in (PSI). C Compiled data from panel B. TPM1, known to undergo SMC specific splicing, was represented in many categories in the rMATS analysis, showing increased usage of exon 2 and reduced usage of exon 3 (D). In panel E, TPM1 protein variants were examined by western blotting showing bands at 36 and 33 kDa that increased relative to other bands. Quantification is included in the bar graph on the right. Panel F shows western blotting for SORBS1. At least five SORBS1 protein species were detected and quantified (right)

Fig 3: Inducible and SMC-specific knockout of Srf in vivo reduces SMC splicing of Vcl, Cald1, and Myocd. To address the in vivo relevance of Myocd-Srf-driven splicing, we generated mice allowing for inducible deletion of Srf in SMCs (Myh11-CreERT2 x Srffl/fl mice injected with tamoxifen, referred to as knockout, KO). Compared to wild type mice (WT), staining for Srf (green), which is a predominately nuclear protein, was reduced in SMCs in the aorta and urinary bladder (A). White lines in the micrographs highlight the SMC layers of the aorta and bladder, respectively. This was confirmed by RT-qPCR, showing reduction of Srf along with three contractile markers in both aorta (B), and bladder (C). We focused on the bladder in view of better Srf depletion and could confirm sizeable reduction of Srf and SMC markers by western blotting (D). Contraction in response to cumulative addition of the muscarinic agonist carbachol (E), depolarization with KCl (60 mM, F), and stimulation with the phosphatase inhibitor Calyculin A (1 µM, G), was reduced, confirming loss of contractility in KO vs. WT bladder. In addition to these expected phenotypes, we also observed a reduction of Rbpms (H), but not of Rbfox2 (I) by RT-qPCR. Splicing of Vcl, Cald1, and Mbnl1 was altered as shown using PCR and agarose gel electrophoresis (J). Myocd splicing (exon 2a) was also examined. Myocd splicing changed from the SMC variant containing exon 2a (+ Ex2a), towards a variant that lacks exon 2a (− Ex2a, panel J, bottom). Silencing of either RBPMS or RBFOX2 similarly favored the heart variant (− Ex2a) of MYOCD in cultured human coronary SMCs (K). Altered splicing of Vcl, and Cald1 in KO bladder was confirmed by western blotting (L)

Fig 4: MYOCD and MRTF-B affect expression of splicing factors. To approach the hypothesis that MYOCD targets splicing factors, we assayed the identified splicing factors at the mRNA level following adenoviral overexpression of myocardin (MYOCD) and the two myocardin family members MRTF-A and MRTF-B. Adenoviruses (200 MOI) were added to human coronary artery SMCs in culture and cells were harvested at 4 days. Transcript levels were determined by RT-qPCR and mRNA fold changes (FC) are shown in (A) (n = 6–12 throughout, error bars represent SEM). B A time-course experiment where Ad-h-MYOCD or Ad-null viruses were added at 0 h and cells were harvested at different times, followed by RT-qPCR for RBPMS (n = 4 null and 4 MYOCD at each time). Panels C and D show western blots for RBPMS (6 days, to account for any delay between mRNA and protein) and RBFOX2 (8 days), respectively. Compiled western blot data is shown below the membranes (n = 10 for C and 6–7 for D). HSP90 was used to ascertain equal protein loading. MYOCD binds to DNA via serum response factor (SRF). To examine the role of SRF, a short hairpin (shSRF) virus was used for knockdown. RBPMS and RBFOX2 were then assayed using RT-qPCR (panel E, coronary artery SMCs under basal conditions, and (F), bladder SMCs transduced with MYOCD, n = 6 throughout). SRF is the positive control. The control construct in this case is referred to as U6 or U6 + MYOCD. SRF knockdown was also combined with MRTF-B overexpression (G), showing that the RBPMS increase with MRTF-B depended on SRF (n = 9). In (E, F), cells were transduced with virus for 8 days, and in (G), cells were transduced for 6 days. Panel H shows the RBPMS gene locus with ChIP-seq data for SRF (green triangles, from the USCS genome browser)

Fig 5: RBPMS RBFOX2 act downstream of MYOCD. A previous study identified a putative splicing complex consisting of RBPMS, RBPMS2, and RBFOX2 (A). We noted that an additional partner in this complex (PICK1) was regulated by MYOCD at the mRNA level, suggesting that MYOCD exerts its splicing effects via 3–4 core partners (green) in a splicing factor complex. Silencing of RBPMS (B), antagonized MYOCD-driven changes in splicing of ACTN1, MBNL1, and VCL as shown using variant-specific primers and agarose gel electrophoresis (C–E, n = 6 in this and the following panels). Similarly, silencing of RBFOX2 (F), antagonized the effect of MYOCD on splicing of these transcripts (G–J). We also examined if MYOCD, but not MRTF-A, can drive differential splicing of vinculin (VCL) using western blotting (K). All MRTFs increased the meta-VCL/VCL ratio (L). RBPMS was preferentially regulated by MYOCD and MRTF-B (M), but RBPMS and the meta-VCL to VCL ratio correlated across the entire dataset (N). All MRTFs similarly promoted VCL splicing in bladder SMCs (O, numbers from the densitometric quantification are shown by the bands). Because MRTF-A and MRTF-B are inhibited by depolymerization of actin, bladder SMCs were treated with Latrunculin B, which depolymerizes actin. This reduced the mRNA levels of RBPMS and RBFOX2 (P) and inhibited SMC-specific splicing (Q, R)