SON and SRRM2 are essential for nuclear speckle formation
- Max Planck Institute for Molecular Genetics, Germany
- Freie Universität Berlin, Germany
Figures
Figure 1 with 2 supplements
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 …
Figure 1—figure supplement 1
SC35 pull-down followed by MS identifies SRRM2 as the top hit and multiple spliceosomal components are co-purified together with SRRM2.
(A) The log of intensities is plotted against detected MS/MS spectra for significantly enriched proteins in the SC35 immunoprecipitations. SRRM2 together with nine most enriched proteins are …
Figure 1—figure supplement 2
A validation for all stable cell lines with transgenic SRSF proteins show only weak localization to the NS.
(A) The domain and size representation of SRSF proteins are shown (left). The inducible stable cell lines are characterized using PAGE (right). The nuclear (N) and cytoplasmic (C) fractions are used …
Figure 2 with 1 supplement
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 …
Figure 2—figure supplement 1
The strategy for making the truncating mutations of SRRM2.
(A) The position of the deepest truncation (tr10) is shown with respect to annotated domains of the protein as well as the dot matrix of the SRRM2 protein sequence. Repetitive regions appear as …
Figure 3 with 1 supplement
SRRM2 truncation#10 leads to loss of SC35 domains but not NS.
(A) SON and RBM25 antibodies are used as NS markers for IF analysis of both SRRM2tr0 and SRRM2tr10 HAP1 cells. No significant impact on the formation of NS in SRRM2tr10 cells in comparison to SRRM2tr…
Figure 3—figure supplement 1
SRSF7 staining in SRRM2tr0 compared to SRRM2tr10 cells.
Immunofluorescence staining of SRSF7 in SRRM2tr0 and SRRM2tr10 cells that are imaged side-by-side show little to no difference in SRSF7 signal in cells that have a truncated SRRM2 that is not …
Figure 4 with 2 supplements
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 …
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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).
- https://cdn.elifesciences.org/articles/60579/elife-60579-fig4-data1-v2.zip
Figure 4—figure supplement 1
SON and SRRM2 are rapidly evolving and largely disordered proteins.
(A) The disorder probability of SON and SRRM2 is shown the same way as in Figure 4B calculated using MobiDB-Lite (up). The same analysis is also carried out on a spliceosomal core protein PRPF8, on …
Figure 4—figure supplement 2
SON and SRRM2 are rapidly evolving and largely disordered proteins.
(A) The disorder probability of SON and SRRM2 is shown similar to Figure 4B and Figure 4—figure supplement 1A calculated using IUPred2A. Both disorder prediction tools resulted in similar plots. (B) …
Figure 5 with 5 supplements
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 …
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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.
- https://cdn.elifesciences.org/articles/60579/elife-60579-fig5-data1-v2.zip
Figure 5—figure supplement 1
Training a machine learning method for detection of NS.
The pipeline used for the quantification of protein localization in NS under various conditions is depicted here. The probability maps generated with ilastik are imported into CellProfiler analysis …
Figure 5—figure supplement 2
Examples of the trained module for detection of NS on different antibody stainings indicate the model predicts NS robustly for each stained protein.
(A) The outcome of the trained model on recognition of NS is shown for SRRM2tr0 HAP1 cells treated with control siRNA (left) or SON siRNA (right). (B) The outcome of the trained model on recognition …
Figure 5—figure supplement 3
Depletion of SON in SRRM2tr10 cells leads to loss of NS but not of Cajal bodies.
(A) PNN 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 PNN is lost upon SON knock-down in …
Figure 5—figure supplement 4
Co-depletion of SON and SRRM2 in SRRM2tr0+GFP HEK293 cells leads to loss of NS.
(A) RBM25 IF signal is shown for four individual cells in each siRNA treatment (control, SRRM2, SON or SRRM2, and SON siRNA) in SRRM2tr0+GFP HEK293 cells. RBM25 signal diffuses out of NS upon SON …
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Figure 5—figure supplement 4—source data 1
Contains ilastik models (.ilp) used to train for nuclear speckles, 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—figure supplement 4.
- https://cdn.elifesciences.org/articles/60579/elife-60579-fig5-figsupp4-data1-v2.zip
Figure 5—figure supplement 5
Co-depletion of SON with RBM25 or SRRM1 in SRRM2tr0 HAP1 cells does not lead to loss of spherical NS.
Knock-down experiments are performed similar to Figure 5—figure supplement 4, but in SRRM2tr0 HAP1 cells, however this time SON is also co-depleted together with RBM25 or SRRM1. Depletion of SON …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | SRRM2 | NCBI | Gene ID: 23524 | |
| Gene (Homo sapiens) | SRSF7 | NCBI | Gene ID: 6432 | |
| Gene (Homo sapiens) | SON | NCBI | Gene ID: 6651 | |
| Cell line (Homo sapiens) | HAP1 | Horizon | Cat. #: C631 | |
| Cell line (Homo sapiens) | Flp-In T-REx HEK293 | Thermo Fisher Scientific | Cat. #: R78007, RRID:CVCL_U427 | |
| Antibody | SC-35 (Mouse monoclonal) | Sigma-Aldrich (Merck) | Cat. #: S4045, RRID:AB_47751 | IF(1:200) WB(1:1000) |
| Antibody | SC-35 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat. #: sc-53518, RRID:AB_671053 | IF(1:100) |
| Antibody | SRRM2 (Rabbit polyclonal) | Thermo Fisher Scientific | Cat. #: PA5-66827, RRID:AB_2665182 | IF(1:100) WB(1:1000) |
| Antibody | SON (Mouse monoclonal) | Santa Cruz Biotechnology | Cat. #: sc-398508 RRID:AB_2868584 | IF(1:100) |
| Antibody | SON (Rabbit polyclonal) | Sigma-Aldrich (Merck) | Cat. #: HPA023535, RRID:AB_1857362 | IF(1:200) WB(1:1000) |
| Antibody | RBM25 (Rabbit polyclonal) | Sigma-Aldrich (Merck) | Cat. #: HPA070713, RRID:AB_2686302 | IF(1:200) WB(1:1000) |
| Antibody | SRRM1 (Rabbit polyclonal) | abcam | Cat. #: ab221061, RRID:AB_2683778 | IF(1:600) WB(1:2000) |
| Antibody | PNN (Rabbit polyclonal) | abcam | Cat. #: ab244250, RRID:AB_2868585 | IF(1:200) WB(1:1000) |
| Antibody | coilin (Rabbit monoclonal) | Cell Signaling | Cat. #: 14168, RRID:AB_2798410 | IF(1:800) WB(1:2000) |
| Antibody | GFP (Rabbit polyclonal) | Chromotek | Cat. #: PAGB1, RRID:AB_2749857 | WB(1:1000) |
| Antibody | SRSF7 (Rabbit polyclonal) | MBL | Cat. #: RN079PW, RRID:AB_11161213 | IF(1:200) WB(1:1000) |
| Antibody | SRSF1 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat. #: sc-33652, RRID:AB_628248 | WB(1:1000) |
| Antibody | SRSF2 (Rabbit monoclonal) | Abcam | Cat. #: ab28428, RRID:AB_777854 | WB(1:1000) |
| Antibody | U1-70K (Mouse monoclonal) | Santa Cruz Biotechnology | Cat. #: sc-390899, RRID:AB_2801569 | WB(1:1000) |
| Antibody | U2AF65 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat. #: sc-53942, RRID:AB_831787 | WB(1:1000) |
| Antibody | DHX9 (Rabbit monoclonal) | abcam | Cat. #: ab183731, RRID:AB_2868586 | WB(1:1000) |
| Antibody | ADAR (Mouse monoclonal) | Santa Cruz Biotechnology | Cat. #: sc-73408, RRID:AB_2222767 | WB(1:1000) |
| Antibody | Tubulin (Mouse monoclonal) | Santa Cruz Biotechnology | Cat. #: sc-32293, RRID:AB_628412 | WB(1:2000) |
| Antibody | Myc (Rabbit monoclonal) | Cell Signaling | Cat. #: 2276, RRID:AB_331783 | WB(1:1000) |
| Antibody | FLAG (Mouse monoclonal) | Sigma-Aldrich (Merck) | Cat. #: F3165, RRID:AB_259529 | IF(1:200) WB(1:2000) |
| Antibody | RNAPII-S2P (Rabbit monoclonal) | Cell Signaling | Cat. #: 13499, RRID:AB_2798238 | WB(1:1000) |
| Recombinant DNA reagent | CRISPaint Gene Tagging Kit | Addgene | Cat. #: 1000000086, RRID:Addgene_1000000086 | |
| Sequence-based reagent | RBM25 siRNA | Thermo Fisher Scientific | Cat. #: s33912 | 10 nM |
| Sequence-based reagent | SRRM1 siRNA | Thermo Fisher Scientific | Cat. #: s20020 | 10 nM |
| Sequence-based reagent | SON siRNA | Thermo Fisher Scientific | Cat. #: s13278 | 10 nM |
| Sequence-based reagent | SRRM2 siRNA | Thermo Fisher Scientific | Cat. #: s24004 | 10 nM |
| Commercial assay or kit | Pierce MS- Compatible Magnetic IP Kit (Protein A/G) | Thermo Fisher Scientific | Cat. #: 90409 | |
| Commercial assay or kit | Lipofectamine RNAiMAX Reagent | Thermo Fisher Scientific | Cat. #: 13778075 | |
| Chemical compound, drug | dTAG-7 | Tocris | Cat. #: 6912 | 1 µM |
| Software, algorithm | ilastik | https://www.ilastik.org/ | RRID:SCR_015246 | |
| Software, algorithm | CellProfiler | https://cellprofiler.org/ | RRID:SCR_007358 | |
| Software, algorithm | Jupyter Lab | https://github.com/ jupyterlab/jupyterlab; Kluyver, 2016 | RRID:SCR_018315 |
Additional files
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Supplementary file 1
The oligos used for: 1. the cloning of SRSF1-12 constructs for making stable-cell lines (shown in Figure 1 and Figure 1—figure supplement 1), 2. guide RNAs used to generate SRRM2 truncation HAP1 cell lines and 3. the genotyping oligos used for characterization of SRRM2 truncation HAP1 cell lines (shown in Figure 2 and Figure 2—figure supplement 1).
- https://cdn.elifesciences.org/articles/60579/elife-60579-supp1-v2.xlsx
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Supplementary file 2
The MaxQuant processed output files showing peptide and protein identification, accession numbers, % sequence coverage of the protein, and q-values.
(shown in Figure 1 and Figure 1—figure supplement 1) are listed.
- https://cdn.elifesciences.org/articles/60579/elife-60579-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/60579/elife-60579-transrepform-v2.docx
