Figure 3: CELF2a favours exon 45 inclusion.
From: The lack of the Celf2a splicing factor converts a Duchenne genotype into a Becker phenotype
(a) GSΔ44 trans-differentiated myocytes were transfected with an empty vector (pcDNA) or with a Flag-CELF2a construct (pFlag-CELF2a-1). Cells were shifted to DM medium for 10 days and the RNA analysed by RT–PCR for Flag-CELF2a expression, for exon 45 skipping (43–46) and for CSDE1 and TMED2 isoforms (upper panel). The expression of the exogenous Flag-CELF2a was tested by western blot with anti-FLAG antibodies (lower panel). GAPDH was used as loading control. (b) Schematic representation of the pLuc45 construct: the DMD gene region encompassing exon 45 plus portions of the flanking introns was cloned into the intron of pcDNA3.1-Luc. This plasmid contains the CMV promoter driving the expression of a Firefly luciferase pre-mRNA that produces luciferase only upon splicing (lower part). (c) The Bar plots show the relative luminescence units (RLU) of Firefly luciferase in control (black bar) and GSΔ44 (grey bar) myoblasts transfected with pLuc45 and incubated 48 h. The values were normalized for Renilla luciferase activity derived from a co-transfected pRL-TK plasmid. Error bars represent standard error from two independent experiments. *P<0.05. (d) The Bar plots show the RLU of Firefly luciferase in HeLa cells transfected with pLuc45 and either pFlag-CELF2a-1 (dotted bar) or pcDNA (dashed bar) and incubated 48 h. The values were normalized for Renilla luciferase activity derived from a co-transfected pRL-TK plasmid. Error bars represent s.e.m. from three independent experiments. **P<0.01. (e) Left panel: schematic representation of part of the dystrophin (DMD) and insulin receptor (IR) pre-mRNAs. Arrows indicate the positions of the oligos used. Right panels: control (CTRL) and GSΔ44 myocytes were UV crosslinked and subjected to nuclear-cytoplasmic fractionation. Nuclear extracts were immunoprecipitated by anti-CELF2 (lane IP) and control IgG antibodies. The presence of the regions indicated on the right (E45, E44–45 and E10 for DMD; IR-I10 for IR) was analysed by RT–PCR. GAPDH was used as negative control. ‘IN’ samples represent 10% of the total nuclear extract. Molecular weight marker: GeneRuler 100 bp (Thermo Scientific). CMV, cytomegalovirus.
