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. 2020 Dec 7;55(5):629–647.e7. doi: 10.1016/j.devcel.2020.09.025

Figure 6.

Figure 6

The SRPK-RNF12 Signaling Pathway Is Deregulated in Human Intellectual Disability

(A) RNF12 WT-KI or R575C-KI mESCs were analyzed for relative mRNA expression by quantitative RT-PCR. Data represented as mean ± SEM (n = 3). Unpaired Student’s t test, two-sided, confidence level 95%. Dll1 (∗∗∗∗) p < 0.0001; Kif1a (∗∗∗) p = 0.0005.

(B) Graphical representation of SRPK intellectual disability variants reported in literature grouped by type of chromosomal mutation (Top) and position within the SRPK3 protein (Bottom).

(C) RNF12 phosphorylation in vitro by WT SRPK3 or the indicated mutants was analyzed by immunoblotting for RNF12 phospho-Ser214 and total RNF12 (Top). Quantification of infrared RNF12 phospho-Ser214 immunoblotting blotting signal normalized to total RNF12 (Bottom). Data represented as mean ± SEM (n = 3). One-way ANOVA followed by Tukey’s multiple comparisons test; confidence level 95%. (∗∗∗∗) p > 0.0001, (∗∗∗) p = 0.0001.

(D) SRPK1, SRPK2, SRPK3, and RNF12 levels in mESCs, hiPSCs (CHiPS4 cell line), and mouse heart lysate were analyzed by immunoblotting.

(E) hiPSC (bubh_3 line) extracts were analyzed for average protein copy number via quantitative proteomics. Data were obtained from the human induced pluripotent stem cell initiative database (http://www.hipsci.org/) and represented as mean ± SEM (n = 24), ND, not detected.

(F) hiPSCs (CHiPS4 cell line) and mESCs were treated with 10 μM SRPKIN-1 for 4 h and RNF12 SR-motif phosphorylation analyzed by phos-tag immunoblotting for RNF12. Fully phosphorylated (4-P) and unphosphorylated (0-P) RNF12 SR-motif is indicated with open (○) and closed (●) circles, respectively.

(G) Single nuclei isolated from post-mortem human brain cortex neurosurgery were analyzed via SMART-seq v4 RNA-seq (data from Hodge et al., 2019). Each bar represents a distinct neuronal sub-type or non-neuronal cell. Trimmed average counts per million (CPM) for SRPK1, SRPK2, and SRPK3 are shown.

(H) Expression of RNF12, SRPK1, SRPK2, and SRPK3 in adult mouse tissues was analyzed by immunoblotting. Ponceau S staining is shown as a loading control.