Fig 2: CARM1 Levels Modulate Paraspeckle Body, Related to Figure 4(A) Scheme for overexpression of wild-type (WT) Carm1 synthetic mRNA. Zygotes were injected with either Carm1-WT synthetic mRNA (50 ng or 100 ng) and Gap43-GFP mRNA only as control. Embryos were cultured until the 4-cell stage and subjected to immunostaining for the indicated markers. (B) Confocal images of CARM1 and p54nrb localization in control embryos and with elevated levels of CARM1 showing a representative nucleus of a 4-cell stage embryo. Yellow arrowheads indicate p54nrb clusters. Scale bar 5 µm. (C) Quantification of p54nrb structures localized around the nucleoli in control and CARM1-WT overexpressing 4-cell stage embryos (10 × 4-cell embryos, 40 nuclei in total per each condition). (D) Quantification of relative fluorescence intensity of CARM1 and p54nrb in control and CARM1-WT overexpressing 4-cell stage embryos (10 × 4-cell embryos, 40 nuclei in total per each condition; Mann-Whitney test, p = 0.5925 and p = 0.7042). (E) Scheme for overexpression of wild-type (WT) Carm1 synthetic mRNA and Gap43-GFP mRNA in one blastomere of 2-cell stage embryo. Embryos were cultured until the 4-cell stage and subjected to immunostaining for the indicated markers. (F) Confocal images of CARM1 and p54nrb localization in control blastomeres and blastomeres with elevated levels of CARM1 (n = 20). Nuclei of a 4-cell stage embryo are shown. Note clustering of p54nrb in lineage of treated cell. Scale bar 5 µm. Error bars represent s.e.m.

Fig 3: CARM1 facilitates Sox2 transactivation activity.(A) The effect of CARM1 knockdown on Sox2 mediated reporter activity assay. FLAG-Sox2 and the luciferase reporter plasmid (8×S/O-Luc) were co-transfected into MCF7 cells with either the plasmid of RNAi vector or siCARM1. (B) The effect of CARM1 overexpression on Sox2-mediated reporter activity. Wild-type Sox2 and its expression vector were individually transfected into MCF7 cells with or without FLAG-CARM1. (C) The effect of AdOx on Sox2-mediated reporter activity. MCF7 cells were first transfected with the 8×S/O-Luc reporter and FLAG-Sox2 for 24 hours. The cells were then treated with different concentrations of AdOx as indicated for another 24 hours before harvest for reporter activity assay. (D) The effect of CARM1-WT and CARM1-R169A overexpression on Sox2-mediated reporter activity. Wild type and truncated Sox2 (as shown in Fig. 1E) and its vector were individually transfected into MCF7 cells with CARM1-WT or CARM1-R169A. Each bar represents mean value ± S.D. from at least three independent experiments. ** P<0.01, Student's t-test.

Fig 4: CARM1 Colocalizes with Paraspeckle Components, Related to Figure 2(A) Segmentation of a single nucleus from the 4-cell stage embryo. Co-immunostaining of CARM1 with paraspeckle component, p54nrb (20 × 4-cell embryos, 80 nuclei in total). Scale bar 10 µm. (B) Proximity of CARM1 and p54nrb analyzed at the 4-cell stage by proximity ligation assay (10 × 4-cell embryos, 40 nuclei in total). Scale bar 10 µm. (C) Immuno-localization of CARM1 and p54nrb with Neat1 RNA detected by in situ hybridization (10 × 4-cell embryos, 40 nuclei in total). Scale bar 5 µm. Error bars represent s.e.m.

Fig 5: Effect of ras/MAPkinase signalling or a phospho-mimetic mutation on the interaction between C/EBPß and PRMT4/CARM1. (A) FLAG-tagged LAP*/C/EBPß1 was co-expressed with HA-tagged PRMT4/CARM1 in QT6 fibroblasts in the absence and presence of rasV12. Immunoprecipitation (IP) from cell lysates with anti-Flag or anti-HA antibody as indicated (top) and protein expression control (below, expr. control). The anti-P-Thr235 antibody specifically reveals C/EBPß that is phosphorylated at the MAPkinase site. IP: immunoprecipitation. IB: immunoblot. (B) Lysates from K562 cells (with/without PMA treatment) were immunoprecipitated with anti-C/EBPß or negative control IgG. Proteins were analysed by immunoblotting with anti-PRMT4/CARM1, anti-C/EBPß, or anti-P-Thr235, as indicated. Protein expression control below (expr. contr.). IP: immunoprecipitation. IB: immunoblot. (C) Relative quantification of phosphorylation and R3me2 on LAP*/C/EBPß1 by multiple-reaction monitoring (MRM). Left: three different tryptic peptides were used for quantification of C/EBPß in total cell lysates from untreated and PMA-treated K562 cells, as indicated. Middle: quantification of the C/EBPß Thr-235 phosphorylation MAPkinase site containing tryptic C/EBPß peptide. Right: quantification of the R3-LAP*/C/EBPß1 methylated tryptic peptide in total cell lysates by MRM. (D) FLAG-tagged LAP*/C/EBPß1 or LAP*/C/EBPß1 T220D constructs were co-expressed with HA-tagged PRMT4/CARM1 in QT6 fibroblasts. Cell lysates were immunoprecipitated with anti-HA and immunoblots (IP) were developed with anti-Flag or anti-HA, respectively (indicated on the right). IP: immunoprecipition. IB: immunoblot. Below: protein expression control (anti-Flag or anti-HA) as indicated. Vertical lines indicate assembly of two separated lanes on the same immunoblot.