Fig 2: 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 3: Schematic diagram showing a proposed model for the interactions among RBFOX2, GOLIM4, and RAB26 in NPC progression. RBFOX2 mediates the alternative splicing of the exon-7 of GOLIM4 through binding to GGAA sequence in the exon, which generates the long isoform of GOLIM4-L. GOLIM4-L promotes tumorigenesis of NPC, likely through vesicle-mediated transport pathway involving recruitment of RAB26.

Fig 4: 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 5: Expression of RBFOX2 in NPC and its association with prognosis of patients with NPC. A) Transcriptome analysis showed the mRNA expression of RBFOX2 in NPC tissues (n = 85) and control samples (n = 10). B) qRT-PCR showed the transcription level of RBFOX2 (in relative to ACTIN) in another independent sample collection of NPC biopsies (n = 20) and control tissues (n = 19). C) Correlation between the PSI of GOLIM4-L and the mRNA level RBOFX2 in samples described in (B). D) Western blotting assays showed the protein expression of RBFOX2 in samples described in (B). ACTIN was used as control. E) Western blotting assays showed the protein levels of RBFOX2 and ACTIN in NPC cell lines and normal nasopharyngeal epithelium cells (NP69). F) Representative images of immunohistochemistry (IHC) assay for formalin-fixed paraffin-embedded NPC tissues (n = 98) with RBFOX2 antibody. Scale bar, 100 µm. G,H) Kaplan–Meier survival curves of overall and disease-free survival in patients with NPC as described in (F) stratified by the protein level of RBFOX2. *p < 0.05, **p < 0.01, ***p < 0.001.