Fig 2: USP42 DUB activity is required for its efficient nuclear speckle localization and phase separation.A, B U2OS cells transfected with GFP-USP42-C120A (catalytically inactive) and GFP-USP42-C truncation expression constructs (domain structures as indicated without GFP tag) were analyzed by immunofluorescence to inspect their colocalization with endogenous SC35. Representative confocal sections were shown with magnified insets. Graphs on the right show quantification. Nucleus was stained with DAPI. Scale bar = 10 μm. C FRAP analysis of the droplets formed by GFP-USP42-C120A in U2OS cells, with relative fluorescence intensities shown in the curve on the right (n = 3). Scale bar = 10 μm.

Fig 3: Immunoprecipitation of Psip1/p52 and p75.A) Immunoblot of NIH 3T3 nuclear extract with antibodies; A300-848 which recognizes only the p75 isoform of Psip1, and A300-847 which detects both p52 and p75. B) IPs with IgG, A300-847 and A300-848 from NIH 3T3 nuclear extracts, immunoblotted with antibodies recognizing p75 (A300-848) or p52 (A300-847). Input is 5% total extract. C) Immunoblot of A300-847 IPs with αSRSF1. IP was also performed in the presence of RNase A. Input is 10% of total extract and IgG served as a control. D) In vitro pulldown of 293T cell expressed T7-SRSF1 using GST-p52 and Psip1-PWWP and immunoblotted with αT7. Input is 5% of T7-SRSF1 and GST alone is control. E) In vitro pulldown with GST-p52 of; T7-SRSF1 and mutants that mimic its hypo-(RG) and hyper-phosporylation (RD), T7-SRSF3 and GFP-SRSF2. Immunoblotting was with αT7 or αGFP. F) ChIP with αH3K36me3 from wild-type (wt) and Psip1 −/− MEFs immunoblotted with antibodies detecting Srsf1, Srsf2, Srsf3, PTB, Psip1 and H3K36me3. G) In vitro pulldown of HeLa core histones by T7-SRSF1 in the presence or absence of Psip1/p52 and immunoblotted with antibodies detecting pan H3, H3K36me3, H3K9me2 and H3K4me3.

Fig 4: Characterization of the GFP-TCERG1 fusion protein. (A) Colocalization of GFP-TCERG1 with the essential splicing factor SRSF2. Dual labeling of HEK293T cells with EGFP-TCERG1 (a, green) and with the SRSF2 antibody (b, red) was performed. DAPI labeling was used to stain the chromatin (c, blue). Merge images are also presented (d). Line scans showing the local intensity distributions of GFP-TCERG1 in green, SRSF2 in red, and DAPI in blue are presented in panel e. A bar in panel d indicates the position of the line scans. Scale bar in panel d, 3 µm. (B) On the left is a schematic representation of the structure of the Bcl-x gene, with exons (boxes) and introns (lines). The two splice variants, the long antiapoptotic Bcl-xL and the short proapoptotic Bcl-xS, which are generated by alternative 5′ splice site selection, are depicted. The dotted lines indicate the alternative splicing events. The effect of TCERG1 and GFP-TCERG1 overexpression on the alternative splicing of the Bcl-x minigene is shown in the middle. HEK293T cells were transfected with the Bcl-x minigene together with empty plasmid (mock, lane 1), TCERG1 (lane 2), or GFP-TCERG1 expression plasmids (lane 3). Cells were harvested and processed for RT-PCR analysis. The graph on the right presents the densitometric analysis results as the ratio of Bcl-xL to Bcl-xS isoforms from three independent experiments (mean ± SEM). (**) P < 0.01; (***) P < 0.001.

Fig 5: Potential significance of PML-NB-free intranuclear environment of oocytes as exemplified by the outcomes of deliberate PML-NBs assembly. As oocytes acquire the developmental competence, chromatin status and their configurations are subjected to drastic alterations that correlate with transcriptional quiescence. Co-localization of SUMO (green) with endogenous PML protein (red) was gradually evident upon meiotic resumption followed by fertilization (normal condition, right side). However, during the maturation of oocytes, well-defined PML clients but not endogenous PML protein co-localized with SUMO not only in the nucleolus but also in peri-centromeric heterochromatin (normal condition, left side). While the selected PML clients remained to stay at peri-centromeric heterochromatin, SUMO committed to the response against proteotoxic stress. SUMO delocalized from peri-centromeric heterochromatin and co-localized with SC35-positive large structures (black) (proteotoxic condition, left side). Microinjection experiments for deliberate assembly of PML-NBs (purple, forming the ‘shell’) revealed that SUMO contributed to the regulation of dynamics as well as prevention of clustering of PML-NBs in the nucleoplasm of oocytes. Under the situation with deliberately assembled PML-NBs, the emergent response of SUMO was not adequately conducted, suggesting that the PML-NB-free intranuclear environment helps reserve SUMO for emergent responses by redirecting the flux of SUMO otherwise needed to maintain PML-NB dynamics.