Journal: bioRxiv

Article Title: Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

doi: 10.1101/2020.04.02.005041

Figure Lengend Snippet: ( A) WT mESCs were treated with either SRPKIN-1 or T3 kinase inhibitors and phosphorylation of Ser-Arg rich splicing factors (SRSF) assessed by immunoblotting. SRPK1 and SRPK2 expression is determined by immunoblotting and ERK1/2 levels are shown as a loading control. (B) Putative SRPK substrates were predicted using the ScanProsite tool and identified proteins grouped according to their UniProt functional description into mRNA splicing-related (tan) and other (cyan). (C) RNF12 domain structure showing phosphorylation sites detected via immunoprecipitation mass-spectrometry. LZL = Leucine-Zipper Like, NLS = Nuclear Localisation Signal, NES = Nuclear Export Signal, RING = RING-type E3 ubiquitin ligase catalytic domain. (D) CMGC family kinase expression in mESCs determined via quantitative total proteomics. Protein copy numbers are represented according to intensity (see right panel) and distributed in the kinase phylogenetic tree using Kinoviewer. (E) Ability of CMGC kinases (200 mU each) to phosphorylate the RNF12 SR-motif in vitro was determined by immunoblotting for RNF12 phospho-Ser214. RNF12 levels are shown as a loading control. (F) mESCs were treated with 10 µM of the following CMGC kinase inhibitors: AZ-191 (DYRK1B inhibitor), KH-CB19 (CLK-DYRK inhibitor), T3 (CLK inhibitor), SPHINX31 (SRPK1 inhibitor), CHIR-99021 (GSK3 inhibitor), PD-0325901 (MEK inhibitor), VX-745 (p38 inhibitor), JNK-IN-8 (JNK inhibitor), RO-3306 (CDK1 inhibitor) and Flavopiridol (CDK7/9 inhibitor) for 4 h and RNF12 SR-motif phosphorylation determined by immunoblotting for RNF12 phospho-Ser214. RNF12 levels are shown as a loading control. (G) SRPK inhibitor dose-response curves for inhibition of RNF12 phosphorylation by SRPK1 and SRPK2 in vitro was determined by immunoblotting for RNF12 phospho-Ser214. (H) SRPKIN-1 inhibition of SRPKs in vivo was determined by pre-treatment of mESCs with 10 µM SRPKIN-1 for 4 h followed by SRPK1 or SRPK2 immunoprecipitation kinase assay using recombinant RNF12 as a substrate. RNF12 SR-motif phosphorylation and SRPK1 or SRPK2 levels analysed by immunoblotting for RNF12 phospho-Ser214. RNF12 levels are shown as a loading control.

Article Snippet: To generate CRISPR Cas9 knockout mESC lines we transfected wild-type (for Srpk1 and Srpk2) or Rlim -/y (for Zfp42) mESCs with pX335 and pKN7 vectors (Addgene) containing gRNA sequences targeting: Srpk1 exon 3, Srpk2 exon 5, Zfp42 exon 4 (detailed in Table S6).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Functional Assay, Immunoprecipitation, Mass Spectrometry, Ubiquitin Proteomics, In Vitro, Inhibition, In Vivo, Kinase Assay, Recombinant

Journal: bioRxiv

Article Title: Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

doi: 10.1101/2020.04.02.005041

Figure Lengend Snippet: (A) Wild-type (WT), Srpk1 -/- and Srpk2 -/- mESCs were cultured and SRPK protein expression was analysed via immunoblotting. Heart, spleen and skeletal muscle tissue lysates and SRPK3 recombinant protein are shown as positive controls for SRPK3 expression. (B) Inhibition of RNF12 phosphorylation in vitro by SRPK1 and SRPK2 in the presence of varying concentrations of the indicated SRPK inhibitors was determined by immunoblotting for RNF12 phospho-Ser214. RNF12 levels are shown as a control

Article Snippet: To generate CRISPR Cas9 knockout mESC lines we transfected wild-type (for Srpk1 and Srpk2) or Rlim -/y (for Zfp42) mESCs with pX335 and pKN7 vectors (Addgene) containing gRNA sequences targeting: Srpk1 exon 3, Srpk2 exon 5, Zfp42 exon 4 (detailed in Table S6).

Techniques: Cell Culture, Expressing, Western Blot, Recombinant, Inhibition, Phospho-proteomics, In Vitro, Control

Journal: bioRxiv

Article Title: Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

doi: 10.1101/2020.04.02.005041

Figure Lengend Snippet: (A) RNF12 knockout ( Rlim -/y ) mESCs were transfected with WT RNF12 or the indicated point mutants and SR-motif phosphorylation analysed by Phos-tag immunoblotting for RNF12. RNF12 4xSA = S212A/S214A/S227A/S229A. RNF12 and ERK1/2 levels are shown as a loading control. (B) RNF12 knockout ( Rlim -/y ) mESCs were transfected with the indicated RNF12 constructs and lysates treated with λ-phosphatase and analysed by Phos-tag immunoblotting for RNF12. Recombinant RNF12 is included as an un-phosphorylated control. (C) mESCs were treated with 10 µM of the of the following CMGC kinase inhibitors: AZ-191 (DYRK1B inhibitor), KH-CB19 (CLK-DYRK inhibitor), T3 (CLK inhibitor), SPHINX31 (SRPK1 inhibitor), CHIR-99021 (GSK3 inhibitor), PD-0325901 (MEK inhibitor), VX-745 (p38 inhibitor), JNK-IN-8 (JNK inhibitor), RO-3306 (CDK1 inhibitor) and Flavopiridol (CDK7/9 inhibitor) for 4 h and RNF12 phosphorylation analysed via Phos-tag immunoblotting for RNF12. RNF12 4xSA is included as an unphosphorylated control. RNF12 and ERK1/2 levels are shown as a control. (D) ESCs were treated with the indicated concentrations of SRPKIN-1 for 4 h and RNF12 phosphorylation analysed via Phos-tag immunoblotting for RNF12. (E) mESCs were treated with 10 µM SRPKIN-1 for 4 h and RNF12 phosphorylation analysed from HA-RNF12 immunoprecipitates via RNF12 phos-tag and phospho-Ser214 immunoblotting using multiplex infrared immunoblot. (F) Phosphorylated peptides detected via mass spectrometry analysis of RNF12 following in vitro phosphorylation by SRPK1. pS=phospho-Serine. (G) Autoradiography of RNF12 wild-type (WT) or S212A/S214A/S227A/S229A (4xSA) following a radioactive kinase reaction with SRPK1, SRPK2 or SRPK3. Coomassie staining of RNF12 protein is shown as a loading control. (H) Srpk1 -/- mESCs were transfected with control or SRPK2 siRNA and RNF12 phosphorylation was analysed via phos-tag immunoblotting. ERK1/2 levels are shown as a loading control.

Article Snippet: To generate CRISPR Cas9 knockout mESC lines we transfected wild-type (for Srpk1 and Srpk2) or Rlim -/y (for Zfp42) mESCs with pX335 and pKN7 vectors (Addgene) containing gRNA sequences targeting: Srpk1 exon 3, Srpk2 exon 5, Zfp42 exon 4 (detailed in Table S6).

Techniques: Knock-Out, Transfection, Phospho-proteomics, Western Blot, Control, Construct, Recombinant, Multiplex Assay, Mass Spectrometry, In Vitro, Autoradiography, Staining

Journal: bioRxiv

Article Title: Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

doi: 10.1101/2020.04.02.005041

Figure Lengend Snippet: (A) HA-RNF12 expressing Rlim -/y mESCs were treated with 10 µM SRPKIN-1 for the indicated times and pSR-motif phosphorylation of HA-immunoprecipitated RNF12 analysed via phos-tag immunoblotting. HA-RNF12 levels are shown as a control (B) SRPKIN-1 activity was profiled in vitro using 50 kinases (MRC-PPU International Centre for Kinase Profiling). Data are represented as mean ± S.D. (n=3). (C) RNF12 expressing mESCs were treated with 10 µM of the following inhibitors: SRPKIN-1 (SRPK inhibitor), CCT-241533 (CHK2 inhibitor), Harmine (DYRK1A inhibitor), WEHI-345 (RIPK2 inhibitor), IRAK-4-Inhibitor-a (IRAK4 inhibitor) and GSK-461364 (PLK1/2 inhibitor) for 4 h, and RNF12 phosphorylation analysed via phos-tag immunoblotting. HA-RNF12 and ERK1/2 levels are shown as a control. (D) RNF12 expressing mESCs were pre-treated with 5 µM SRPKIN-1 for 3 h, media changed and cells cultured for further 5 h (+ wash-out). RNF12 phosphorylation was analysed via Phos-tag gels. ERK1/2 levels are shown as a loading control. (E) Multiple Srpk1 -/- and Srpk2 -/- mESC clones were analysed for RNF12 phosphorylation via phos-tag immunoblotting. SRPK, RNF12 and ERK1/2 levels are shown as controls.

Article Snippet: To generate CRISPR Cas9 knockout mESC lines we transfected wild-type (for Srpk1 and Srpk2) or Rlim -/y (for Zfp42) mESCs with pX335 and pKN7 vectors (Addgene) containing gRNA sequences targeting: Srpk1 exon 3, Srpk2 exon 5, Zfp42 exon 4 (detailed in Table S6).

Techniques: Expressing, Phospho-proteomics, Immunoprecipitation, Western Blot, Control, Activity Assay, In Vitro, Cell Culture, Clone Assay

Journal: bioRxiv

Article Title: Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

doi: 10.1101/2020.04.02.005041

Figure Lengend Snippet: (A) Subcellular localisation of RNF12 wild-type knock-in (WT-KI), SR-motif phosphorylation site knock-in (4xSA-KI) or SR-motif deletion (ΔSR-KI) in mESCs was determined by immunofluorescence. Scalebar: 20 µm. (B) RNF12 WT-KI or 4xSA-KI mESCs were treated with 30 nM Leptomycin B for 6 h and RNF12 localisation analysed via immunofluorescence. Scalebar: 20 µm. (C) FLAG-tagged SRPK1 and SRPK2 were transfected in mESCs and localisation of SRPKs and RNF12 analysed via immunofluorescence. Scalebar: 20 µm. (D) REX1 steady-state levels were analysed in RNF12 WT-KI, 4xSA-KI, ΔSR-KI and W576Y-KI mESCs by immunoprecipitation followed by immunoblotting. RNF12 and ERK1/2 levels are shown as a control. (E) REX1 half-life was determined in RNF12 WT-KI, 4xSA-KI, ΔSR-KI and W576Y-KI mESCs by immunoblotting. ERK1/2 levels are shown as a loading control.

Article Snippet: To generate CRISPR Cas9 knockout mESC lines we transfected wild-type (for Srpk1 and Srpk2) or Rlim -/y (for Zfp42) mESCs with pX335 and pKN7 vectors (Addgene) containing gRNA sequences targeting: Srpk1 exon 3, Srpk2 exon 5, Zfp42 exon 4 (detailed in Table S6).

Techniques: Knock-In, Phospho-proteomics, Immunofluorescence, Transfection, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

doi: 10.1101/2020.04.02.005041

Figure Lengend Snippet: (A) Time course of RNF12 in vitro phosphorylation by SRPK1 analysed for SR-motif phosphorylation via pSer214 infrared and Phos-tag immunoblotting. RNF12 levels are shown as a control. (B) RNF12 in vitro phosphorylation by SRPK2 for 1 h was analysed via multiplex infrared Phos-tag and regular immunoblotting. This material is representative of samples used in E2 ubiquitin discharge assays displayed in . (C) Recombinant RNF12 was incubated with SRPK1 in absence or presence of SRPKIN-1 and subjected to REX1 fluorescent ubiquitylation assays. Infrared scans of ubiquitylated substrate signal and graphical quantifications are shown. One-way ANOVA followed by Tukey’s multiple comparisons test; confidence level 95%. (*) P=0.0221 (n=3). Phosphorylated and total RNF12, REX1 and SRPK2 infrared immunoblots are showed as controls. (D) Recombinant RNF12 was incubated with wild-type (WT) or kinase dead (KD) SRPK1 and subjected to REX1 fluorescent ubiquitylation assays. Infrared scans of ubiquitylated substrate signal and quantifications (right panel) are shown. One-way ANOVA followed by Tukey’s multiple comparisons test; confidence level 95%. (***) P=0.0002 (n=3). Phosphorylated and total RNF12, REX1 and SRPK1 infrared immunoblots are showed as controls.

Article Snippet: To generate CRISPR Cas9 knockout mESC lines we transfected wild-type (for Srpk1 and Srpk2) or Rlim -/y (for Zfp42) mESCs with pX335 and pKN7 vectors (Addgene) containing gRNA sequences targeting: Srpk1 exon 3, Srpk2 exon 5, Zfp42 exon 4 (detailed in Table S6).

Techniques: In Vitro, Phospho-proteomics, Western Blot, Control, Multiplex Assay, Ubiquitin Proteomics, Recombinant, Incubation

Journal: bioRxiv

Article Title: Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

doi: 10.1101/2020.04.02.005041

Figure Lengend Snippet: (A) Recombinant RNF12 was incubated with SRPK2 in the absence or presence of 10 µM SRPKIN-1 and subjected to REX1 fluorescent ubiquitylation assays. Infrared scans of ubiquitylated substrate signal and quantifications are shown. Data are represented as mean ± S.E.M. (n=3). One-way ANOVA followed by Tukey’s multiple comparisons test; confidence level 95%. (*) P=0.0350, (****) P<0.0001. Phosphorylated and total RNF12, REX1 and SRPK2 infrared immunoblots are showed as controls. * = non-specific fluorescent signal. (B) Recombinant RNF12 was incubated with wild-type (WT) or kinase dead (KD) SRPK2 and subjected to REX1 fluorescent ubiquitylation assays. Infrared scans of ubiquitylated substrate signal and quantifications are shown. Data are represented as mean ± S.E.M. (n=3). One-way ANOVA followed by Tukey’s multiple comparisons test; confidence level 95%. (*) P=0.0350, (****) P<0.0001. Phosphorylated and total RNF12, REX1 and SRPK2 infrared immunoblots are showed as controls. * = non-specific fluorescent signal. (C) Recombinant RNF12 was incubated with wild-type (WT) or kinase dead (KD) SRPK2 and subjected to SMAD7 fluorescent ubiquitylation assays. Infrared scans of ubiquitylated substrate signal is shown. Phosphorylated and total RNF12, REX1 and SRPK2 infrared immunoblots are showed as controls. * = non-specific fluorescent signal. (D) Recombinant RNF12 was incubated with WT or KD SRPK2 and subjected to E2 ubiquitin discharge assay for the indicated times. Infrared immunoblot scans (top) and reaction rate determinations are shown (bottom). Data are represented as mean ± S.E.M. (n=3). One-way ANOVA followed by Tukey’s multiple comparisons test; confidence level 95%. (*) P=0.0490. Phosphorylated and total RNF12, REX1 and SRPK2 infrared immunoblots are showed as controls.

Article Snippet: To generate CRISPR Cas9 knockout mESC lines we transfected wild-type (for Srpk1 and Srpk2) or Rlim -/y (for Zfp42) mESCs with pX335 and pKN7 vectors (Addgene) containing gRNA sequences targeting: Srpk1 exon 3, Srpk2 exon 5, Zfp42 exon 4 (detailed in Table S6).

Techniques: Recombinant, Incubation, Western Blot, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

doi: 10.1101/2020.04.02.005041

Figure Lengend Snippet: (A) RNF12 WT-KI or R575C-KI mESC lines were analysed for relative mRNA expression of the indicated genes via quantitative RT-PCR. Data are represented as mean ± S.E.M. (n=3). Unpaired Student’s t test, two-sided, confidence level 95%. Dll1 (****) P<0.0001; Kif1a (***) P=0.0005. Gapdh was used as housekeeping control. (B) Graphical representation of the number of SRPK mutations reported in literature grouped by type of chromosomal mutation (Top). Domain structure of SRPK3 indicating localisation of intellectual disability mutations (Bottom). (C) RNF12 phosphorylation in vitro by wild-type (WT) SRPK3 or the indicated mutants was analysed by immunoblotting for RNF12 phospho-Ser214. Total RNF12 and SRPK3 levels are shown as control (Left). Quantification of infrared RNF12 phospho-Ser214 immunoblotting blotting signal normalised to total RNF12 (Right). Data are represented as mean ± S.E.M. (n=3). One-way ANOVA followed by Tukey’s multiple comparisons test; confidence level 95%. (****) P>0.0001, (***) P=0.0001. (D) Expression of RNF12, SRPK1, SRPK2 and SRPK3 in adult mouse tissues analysed via immunoblotting. Ponceau S staining is shown as a loading control. (E) Primary cortical neurons isolated from E16.5 C57BL6 mice were cultured in vitro for the indicated number of days and RNF12, SRPK1 and SRPK2 protein expression analysed via immunoblotting alongside indicated mESC lines. Synaptophysin and Actin expression shown as neuronal maturation marker and loading control respectively. (F) Primary cortical neurons isolated from E16.5 C57BL6 mice were cultured in vitro for the indicated number of days and analysed by immunofluorescence. MAP2 staining is shown as neuron specific marker and Hoescht used as DNA marker. Scalebar: 20 µm. (G) RNF12 phosphorylation during in vitro mouse cortical neuron maturation was analysed via phos-tag immunoblotting. Synaptophysin and Actin expression are shown as neuron maturation marker and loading control respectively. (H) Cortical neurons were cultured for 21 days and treated with 10 µM SRPKIN-1 for 4 h whereupon RNF12 phosphorylation was analysed via phos-tag immunoblotting. Synaptophysin and Actin expression shown as neuronal maturation marker and loading control respectively.

Article Snippet: To generate CRISPR Cas9 knockout mESC lines we transfected wild-type (for Srpk1 and Srpk2) or Rlim -/y (for Zfp42) mESCs with pX335 and pKN7 vectors (Addgene) containing gRNA sequences targeting: Srpk1 exon 3, Srpk2 exon 5, Zfp42 exon 4 (detailed in Table S6).

Techniques: Expressing, Quantitative RT-PCR, Control, Mutagenesis, Phospho-proteomics, In Vitro, Western Blot, Staining, Isolation, Cell Culture, Marker, Immunofluorescence