Fig 1: SON and SRRM2 form NS in human cells.(A) RBM25 IF signal is shown for four individual cells in each siRNA treatment (control or SON siRNA) in SRRM2tr0 and SRRM2tr10 HAP1 cells. The NS localization of RBM25 is severely reduced upon SON knock-down in SRRM2tr0 cells, and completely lost upon SON knock-down in SRRM2tr10 cells. The quantification of the RBM25 signal within the nucleus is plotted against the RBM25 signal within NS (right panel) using ilastik to train detection of NS and CellProfiler for quantification on 10 imaged fields with a 63X objective (in SRRM2tr0 cells control n = 329, SON-KD n = 422; in SRRM2tr10 cells control n = 329, SON-KD n = 402). Each circle represents a cell and the size of the circles is proportionate to the signal intensity of SON. Inset shows the distribution of the ratio of signal detected in NS over signal detected in the nucleus of each cell. (B) SRRM1 IF signal is shown for four individual cells in each siRNA treatment (control or SON siRNA) in SRRM2tr0 and SRRM2tr10 HAP1 cells. The NS localization of SRRM1 is reduced in SON knock-down in SRRM2tr10 cells and lost upon SON knock-down in SRRM2tr10 cells. The quantification of the SRRM1 signal within the nucleus is plotted against the SRRM1 signal within NS (right panel) using ilastik to train detection of NS and CellProfiler for quantification on 10 imaged fields with 63X objective (in SRRM2tr0 cells control n = 494, SON-KD n = 229; in SRRM2tr10 cells control n = 225, SON-KD n = 247). Inset shows the distribution of the ratio of signal detected in NS over signal detected in the nucleus of each cell. (C) Distribution plots showing the ratio of signal detected in NS over signal detected in the nucleus of each cell, in each condition. The dashed line indicates the median ratio in each condition. See Figure 5—figure supplement 3A for a full version of this analysis for PNN. Scale bars = 5 µm.Figure 5—source data 1.Contains two folders, ‘NS’ and ‘Cajal’.Both folders contain ilastik models (.ilp) used to train for nuclear speckles and Cajal bodies and Cell Profiler pipelines (.cpproj) used to process the probability maps generated by ilastik. The outputs from Cell Profiler (.csv) files are used to generate the figures in the Jupyter Lab environment (.ipynb), shown in Figure 5 and Figure 5—figure supplement 3 respectively.
Fig 2: Splicing factor condensates occur at active super-enhancer-driven genesa. Representative images exhibiting overlap between IF of splicing factors SRSF2, SF3B1, U2AF2, HNRNPA1, SRSF1, SRRM1, PRPF8, or SNRNP70 with nascent RNA FISH of Nanog in fixed mESCs. The rightmost column shows average IF signal for splicing factors centered on randomly selected nuclear positions (see methods).b. Representative lattice light sheet images of live mESCs engineered to express GFP tagged SRSF2 and Halo-JF646 tagged Pol II. Maximum intensity projection after background subtraction.c. Representative images exhibiting overlap or absence of overlap between IF of SRSF2 and DNA FISH of Trim28 in mESCs treated with DMSO or splicing inhibitor Pladienolide B for 4 hrs.
Fig 3: Splicing factors at Trim28, SRSF2/Pol II molecule quantification.a. Representative images exhibiting overlap between IF of splicing factors SRSF2, SF3B1, U2AF2, HNRNPA1, SRSF1, SRRM1, PRPF8, or SNRNP70 with nascent RNA FISH of Trim28 in fixed mESCs.b. Left: Histogram of the integrated intensity of single Halo-JF646 (n=178) and single GFP emitters (n=177). Mean values of 164.8 +/- 5.6 cts (mean +/- s.e.m.) and 108.6 +/- 5.1 (mean +/- s.e.m.) were used to normalize the integrated intensity of Pol II-Halo-JF646 and SRSF2-GFP, respectively. Right: Scatter plot depicting estimated numbers of Pol II and SRSF2 molecules in colocalizing Pol II and SRSF2 puncta (see methods).
Fig 4: SON and SRRM2 are rapidly evolving and largely disordered proteins.(A) The size distribution of SON and SRRM2 is highly variable across metazoan species with a mean length of 2227.9 a.a. and SD of 1149.5 for SON and a mean length of 1928.6 a.a. and SD of 919.3 for SRRM2. The lengths of other NS-associated proteins are less variable with a mean length of 895.1 a.a and SD of 104.2 for SRRM1; mean length of 835.4 a.a. and SD of 77.9 for RBM25; mean length of 652.8 a.a. and SD of 118.7 for PNN, mean length of 2332.8 a.a. SD of 40.8 for PRP8. (B) The disorder probability of SON and SRRM2 is predicted using the MobiDB-Lite algorithm, which shows an increase of disordered content with the increase of protein length for SRRM2, and to some extent, SON. The SON and SRRM2 graphs plotted side-by-side do not correspond to the same species, for a phylogeny resolved version of this graph see Figure 4—figure supplement 1 and for the alternative algorithm (IUPred2A) see Figure 4—figure supplement 2. The color is scaled from dark blue to yellow indicating a decrease in order as the value approaches 1.0 (yellow).Figure 4—source data 1.Contains the numerical values of protein lengths shown in Figure 4A and disorder predictions shown in Figure 4B and Figure 4—figure supplements 1 and 2 (.csv) using two alternative algorithms (IUPred2A and MobiDB-Lite).
Fig 5: 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).
Supplier Page from Abcam for Anti-SRRM1 antibody