Fig. 6: Srsf3 downregulation compensates for HNRNPU loss of activity.

A Images of E14 derived mouse cortex neurospheres electroporated with either px330 (CRISPR/CAS9 Control plasmid) or with two sgRNA sequences, for either Hnrnpu, Tp53, or Srsf3. When genes are targeted (Hnrnpu and Tp53 or Hrnrnpu and Srsf3), neurospheres grow to large sizes (DAPI, Blue) and incorporate EdU (Magenta) despite the accumulation of activated Caspase 3 (CC3, green). B Quantification of the size of neurospheres, two days post electroporation treated with the indicated combination of CRISPR/CAS9 sgRNA’s or overexpressing MDM2. Px330, n = 10 Hnrnpu sgRNA n = 12 Hnrnpu + Tp53 sgRNA n = 13 Hnrnpu + srsf3 sgRNA n = 11 Hnrnpu sgRNA +MDM2 n = 12 Tp53 sgRNA n = 11 Srsf3 sgRNA n = 12 MDM2 n = 12. Error bars ±SE. Analysis was done using ordinary one-way ANOVA with Dunnett’s multiple comparisons test. C Splice variants sensitive PCR of cDNA prepared from treated neurospheres (depicted in panels A and B) shows correction of the over-representation of MDM2 Exon 3 skipping splice variant (300 bp vs. 250 pb, actin band size: 150 bp). One-way ANOVA with Dunnett’s multiple comparison test. Adjusted p values vs. control: Hnrnpu < 0.0001, Hnrnpu+Tp53, Hnrnpu+ Srsf3 NS, Hnnrpu + MDM2 NS, Tp53 p = 0.0007, Srsf3 p = 0.0005, MDM2 p < 0.0001. Error bars (±SEM). D qPCR of the relative abundance of MDM2 isoforms in cultured Neurospheres (NS). Graphs depicted transcript levels, compared to control (p x 330, no sgRNA) of MDM2 splice variants (MDM2 lacking exon 3 (MDM2ΔE3)/MDM2 with retained E3 (MDM2) in NS treated with the indicated combination of CRISPR/CAS9 sgRNA’s, n = 6. Error bars (±SEM) are indicated. Coronal sections (60 μM) shows the location of radially migrating cortical neurons electroporated with the control plasmid (blue), Hnrnpu sgRNA (red), Srsf3 sgRNA (green), both genes (purple). E, F Distribution GFP + cells (%) in five horizontal bins across the cortex width (1 the most apical), Averages, and error bars (±SEM). VZ-Ventricular Zone, CP-cortical plate. Three biological repeats were used; 2–4 70 μM thick slices of comparable position were analyzed of each electroporated brain. Control n = 8, Hnrnpu sgRNA n = 11, Srsf3 sgRNA n = 9, Srsf3 + Hnrnpu sgRNA n = 7. Analysis was done using two-way ANOVA with Tukey’s multiple comparisons test. P values: **p < 0.01, ***p < 0.001, ****p < 0.0001, ns non significant. Source data are provided as a Source Data file.