Figure 7.

The SRPK-RNF12 Signaling Pathway Operates in Neurons

(A) Primary cortical neurons isolated from E16.5 C57BL6 mice were cultured for the indicated number of days in vitro (DIV) and RNF12, SRPK1, and SRPK2, synaptophysin and actin levels analyzed via immunoblotting alongside the indicated mESC lines.

(B) Cortical neurons were cultured for 21 days and treated with 10 μM MG132 and protein levels analyzed by immunoprecipitation and immunoblotting (REX1) and immunoblotting (RNF12 and ERK1/2).

(C) Cortical neurons were cultured in vitro for the indicated number of days (DIV) and RNF12 and MAP2 neuron specific marker analyzed by immunofluorescence. Scale bar: 20 μm.

(D) RNF12 SR-motif phosphorylation during in vitro mouse cortical neuron maturation was analyzed via phos-tag immunoblotting for RNF12. Fully phosphorylated (4-P) and unphosphorylated (0-P) RNF12 SR-motifs are indicated by open (○) and closed (●) circles, respectively. Synaptophysin and actin levels were determined by immunoblotting.

(E) Cortical neurons were cultured for 21 days and treated with 10 μM SRPKIN-1 for 4 h RNF12 SR-motif phosphorylation was analyzed by phos-tag immunoblotting for RNF12. Fully phosphorylated (4-P) and unphosphorylated (0-P) RNF12 SR-motif is indicated with open (○) by closed (●) circles, respectively. Synaptophysin and actin levels were determined by immunoblotting.

(F) The SRPK-RNF12-REX1 signaling pathway regulates neural gene expression and is disrupted in intellectual disability disorders. SRPK phosphorylates the RNF12 SR-motif to promote REX1 ubiquitylation and proteasomal degradation, which acts as a “brake” for neural gene expression in self-renewing pluripotent stem cells. In intellectual disability, inactivating mutations in SRPKs or RNF12 lead to REX1 accumulation and aberrant induction of neural genes.