Fig 2: SC35 mAb immunoprecipitation followed by MS identifies SRRM2 as the top hit.(A) The Top50 hits identified by the MS are depicted on a heatmap showing the number of unique peptides detected for each protein. Also see Figure 1—figure supplement 1A for an intensity vs MS/MS spectra plot and Supplementary file 2 for raw MaxQuant results (B) Streptavidin pull-down of biotin tagged ectopically expressed SRSF proteins 1 to 12 are blotted with FLAG antibody in order to show the amounts of loaded proteins on PAGE. Western blot using mAb SC35 detects SRSF7 with highest sensitivity in comparison to all other SRSF proteins, but also weakly reacts with SRSF1, 2, and 12. Specific antibodies against SRSF2 and SRSF7 are used to validate the authenticity of the purified proteins from stable cell lines in corresponding lanes and blots. (C) SC35-IP performed on lysates from wild-type HEK293 cells identifies SRRM2 as the most enriched protein with a weaker enrichment for SRSF7 but no enrichment for SRSF1 or SRSF2 using western blotting (compare Lane 4 across blots). (D) Homozygous knock-in of the 2xMyc-FKBP12F36V tag into SRSF7 gene locus shifts the SC35 band from 35 kDa to 50 kDa (compare Lanes 1 and 3 on the SC35 blot) and upon induction of degradation with dTAG7 the shifted band is lost (compare Lanes 3 and 4 in Myc, SRSF7, and SC35 blots). This blot validates that the 35 kDa band identified by mAb SC35 blots corresponds to SRSF7.

Fig 3: Endogenous truncating mutations of SRRM2 prove mAb SC35 as an SRRM2 antibody.(A) The strategy for the CRISPaint generated endogenous truncating mutations (0-to-10) accompanied by the TagGFP2 (depicted as GFP for simplicity) fusion are shown. (B) The sizes of SRRM2 truncated GFP fusion proteins are displayed. (C) Protein purified using a GFP-trap pull-down from lysates of corresponding stable HAP1 cell lines carrying the truncated SRRM2 alleles are run on PAGE. Western blotting of SRRM2 using an antibody generated against the common N-terminus is used to show the amount of loaded protein on the gel. SC35 blot shows a significant reduction in signal intensity of SRRM2-tr5 and a complete loss of signal from SRRM2-tr6 to tr10. (D) GFP-trap pull-down performed on lysates from wild-type, tr0 and tr10 HAP1 cells enrich for SRRM2 in tr0 cells, indicating the GFP-tagged allele is specific to SRRM2 and is detected by also SC35 blot (Lanes 1 and 2 inputs compared to Lanes 5 and 6 on the upper and lower left-side blots). SRRM2 also co-purifies two other NS-associated proteins; SRRM1 and RBM25 (Lane 6 on lower right-side blots). SRRM2-tr10 is not detected by SC35 but the pull-down efficiency (Lane 7 on upper left-side blot) and loading is validated by SRRM2 (Lanes 3 and 7 on lower left-side blot) and GFP blots (Lanes 3 and 7 on upper left-side blot). (E) Total cell lysates from wild-type, tr0 and tr10 HAP1 cells are run on 4–12% polyacrylamide gel and blotted with SC35 reveal the high-molecular weight (~300 kDa) as the most intense band and the absence of signal in tr10 cell lines validates that this band represents SRRM2 (filled arrow head). Longer exposure of the blot reveals a weak cross-reactivity with a 35 kDa protein, most likely to be SRSF7, around 35 kDa (empty arrow head).

Fig 4: Localization of SANS in nuclear speckles and Cajal bodies. (A) Immunofluorescence staining of SANS (green) and the nuclear speckle marker/spliceosomal protein SC35 (red) in nuclei of HEK293T cells counterstained with DAPI (blue). (A′) Intensity plots for the regions of interest in A (white arrows) indicate co-localization of SANS and SC35 in nuclear speckles (asterisks). Blue vertical dashed lines indicate nuclear extensions. Positive Pearson′s correlation coefficient (R) supports co-localizations of both proteins. (B) 3D-STED microscopy images of SANS and SC35 co-stained speckles. XY and YZ axes demonstrate close association of SANS with SC35 in nuclear speckles. (C) Immunofluorescence of SC35 (red) in siRNA-treated HEK293T cells counterstained with DAPI (blue). Nuclear speckles size increases in SANS-depleted cells (siSANS) comparted with control siRNA-treated cells (siCtrl). (D) Double immunofluorescence of SANS (RpAb, green) and Coilin (red), a molecular marker for Cajal bodies in HEK293T cells counterstained with DAPI (blue). (D′) Fluorescence intensity plots for region of interest indicated by arrow in D reveals SANS localization at the periphery of Cajal bodies in nuclei. (E) 3D-STED microscopy images of SANS (RpAb, green) and Coilin (red). XY and YZ axes demonstrate localization of SANS at the periphery of Cajal bodies. (F) Immunofluorescence of Cajal bodies by anti-Coilin (red) in SANS-depleted HEK293T cells. (F′) Quantification of the number of Cajal bodies (CBs) in SANS-depleted cells; graph shows the percentage of cells with 0–1, 2 or more CBs. SANS depletion increases the number of Cajal bodies in nuclei (In 4 independent experiments 200 and 160 cells were counted in siCtrl and siSANS treated cells, respectively.). ***P values < 0.001.

Fig 5: SANS depletion blocks release of tri-snRNP complexes from Cajal bodies and recruitment of mature tri-snRNP complexes to nuclear speckles. (A) Proteomic analysis of precipitated tri-snRNP complexes via FLAG-PRPF4 from nuclear extracts of control cells and SANS-depleted cells. Graph displays peptide spectral counts for each protein in logarithmic scale. Green: tri-snRNPs; red: coilin (COIL) and SC35-interacting protein (SCAF11). There was no significant change in the composition of the tri-snRNP in SANS-depleted nuclear extract, but the tri-snRNP was largely bound to the Cajal body scaffold protein coilin. (B) Western blot analysis of immunoprecipitation of intrinsic PRPF4 from lysates of HEK293T cells demonstrates stronger interactions of the tri-snRNP protein PRPF4 with coilin in SANS-depleted cells. SANS depletion does not affect tri-snRNP complex formation visualized by PRPF4 and PRPF6 interaction. (C) Double immunofluorescence staining reveals reduced PRPF31 staining in SC35 positive nuclear speckles in SANS-depleted cells indicating a defective recruitment of the tri-snRNP complex to nuclear speckles. (C′) Quantification of mean immunofluorescence intensity ratios for PRPF31, SON and SF3B1 observed in nuclear speckles and for PRPF31 in Cajal bodies (CB) versus nucleus. (D) Quantifications of PLA signals for SON-, SF3B1- and PRPF31-SC35 show an increase of SON and SF3B1, but a decrease of PRPF31 in nuclear speckles of SANS-depleted cells. (D′) Quantification of PLA signals for PRPF31-Coilin reveals the accumulation of PRPF31 in Cajal bodies of SANS-depleted cells. ***P values < 0.001, *P values < 0.05.