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. 2020 Mar 9;11:1270. doi: 10.1038/s41467-020-15003-7

Fig. 4. RNF113 is a RNA-binding protein.

Fig. 4

a, b RNF113A is recruited on RNAs. Unstimulated or Cisplatin-treated A549 cells were treated with paraformaldehyde to fix larger complexes (a) or irradiated with UV to covalently crosslink direct RNA-protein interaction (b), incubated or not with RNase H and RNA immunoprecipitations using OligodT magnetic beads were done followed by WB analyses (left lanes). Cell extracts before RNA Immunoprecipitations were also subjected to WB analyses (“Input”). c RNF113A-depleted lung cancer cells accumulate R-loops. Control or RNF113A-depleted A549 cells were subjected to immunofluorescence analyses using the anti-R-loops antibody (63X objective lens). A quantification of DNA-RNA hybrids is illustrated. For quantification, nuclei specles in a total of 50 nucleus in each experimental condition were counted (Student t-test, ***p < 0.001). d RNF113A promotes SF3B2, Rad51 and RNF8 expression. Control or RNF113A-depleted A549 cells were untreated or stimulated with Cisplatin and WB analyses were done. e, f RNF113A is required for the integrity of the spliceosome. Control or RNF113A-depleted A549 (e) or BZR-T33 (f) cells treated or not with Cisplatin were subjected to anti-IgG (negative control) or -SF3B1 immunoprecipitations followed by anti-SF3B2 WBs. Cell extracts were also subjected to WBs (lower panels). g SF3B2-depleted lung cancer cells accumulate DNA-RNA hybrids. Control or SF3B2-depleted A549 cells were subjected to immunofluorescence analyses as described in (c). h SF3B2 deficiency enhances DNA-PKcs phosphorylation upon Cisplatin treatment. Control and SF3B2-depleted A549 cells were treated or not with Cisplatin (25 μM for 24 h) and WB analyses were done. i SF3B2 deficiency enhances cell death upon DNA damage. On the left, cell survival upon Cisplatin treatment in control and SF3B2-depleted A549 cells was assessed by FACS. The percentage of cells in early or late apoptosis is quantified. On the right, FACS data from two independent experiments are illustrated (Student t-test, ***p < 0.001). j SF3B1, SF3B2, and RNF113A moves from the nucleus to the cytoplasm of lung cancer cells upon DNA damage. A549 cells were treated or not with Cisplatin and WB analyses were done (cytoplasmic and nuclear extracts).