Fig. 7: RBM4 regulates Hsf1 intron excision via a CU-rich motif and antagonizes the negative effect of PTBP1/2.

A Schematic of the Hsf1 minigene spanning exon 6 to exon 8 of mouse Hsf1. SV40 denotes the promoter. B The Hsf1 minigene and the Rbm4 shRNA (left panel) or FLAG-RBM4 (right panel) vectors were co-transfected into GCs. RT-PCR was performed to detect intron 6 retention/excision. PIR (%) was indicated below the gels (n = 3); PIR measurement was the same in the following panels. Bar graphs show relative PIR of introns 6/7; con shRNA or empty vector was set to 1. C The Hsf1 minigene was transfected into GCs followed by NMDA or DCS treatment. Bar graph for relative PIR is shown as in (B). D Diagram shows the mutant Hsf1, of which nucleotides 3–6 of exon 7 were mutated. GCs were transfected with the wild-type or mutant minigene together with the empty or FLAG-RBM4 vector (left panel) or followed by NMDA treatment (right panel). E The Hsf1 minigene was co-transfected with the empty vector (vec) or vector expressing RBM4, PTBP1, or PTBP2. F The Hsf1 minigene was transfected with the vector of PTBP1 or PTBP2 alone (lane 2) or together with increasing amounts of the RBM4 vector (lanes 3–5). Lane 1 contained the Hsf1 minigene only. Model shows that RBM4 and PTB proteins may competitively regulate Hsf1 intron 6 splicing via a CU-rich motif. G Conclusion model: Rbm4 knockout caused IR of Hsf1 and hence reduced HSF1 protein level. In vitro results demonstrated that RBM4 promotes intron excision of Hsf1 via a CU-rich element and hence increased HSF1 and subsequent BDNF expression, and that NMDAR signaling potentiates RBM4 in splicing regulation. In vivo, evidence revealed that HSF1 rescues the foliation defects and motor learning ability of the Rbm4dKO cerebellum.