Figure 8.

Rbm24 regulates AS to modulate the myogenic process. (A) Potential Rbm24 binding motif analyses and scheme of minigene construction of Mef2d, Rock2 and Lrrfip1. For Lrrfip1, potential Rbm24 binding sites were found 274 bp downstream of Exon 16, and 198 bp upstream and 125 bp downstream of Exon 17. For Mef2d, potential Rbm24 binding sites were found at 8 bp upstream of Exon α1 and 33 bp downstream of Exon α2. For Rock2, potential Rbm24 binding sites were found 120 bp downstream of Exon 28. (B) RT-PCR analysis of AS of minigene in response to Rbm24 overexpression. The GFP expression plasmid was used as a negative control. Green boxes, flanking constitutive exons; blue and orange boxes, alternative exons. The primers are indicated by arrowheads. (C) Diagram showing the scheme of construction of C2C12 cell lines in which Rbm24 expression was knocked down and different isoforms of Mef2d were overexpressed. (D) RT-PCR (Top panel) and western blot (Bottom panel) results showing successful overexpression of Mef2dα1 (undifferentiated isoform) and Mef2dα2 (differentiated isoform), and the knockdown of Rbm24 expression. Cells were treated with lentivirus expressing scrambled shRNA or Rbm24 shRNA (shRbm24), or lentivirus expressing either flag-tagged Mef2dα1 or Mef2dα2. Total RNAs and proteins were extracted from C2C12 3 days after switching to the differentiated medium. (E) Immunostaining analysis of MyHC-positive cells after 3 days of differentiation. Scale bars, 100 µm. (F) Statistical analysis of the MyHC-positive cells in (E). At least four different visual fields were analyzed to calculate the percentage of MyHC-positive cells in each group. Data are presented as mean ± SEM, **P < 0.01, unpaired t-test.