Fig. 5

ScaRNA2 bridges RPA-ssDNA to the assembly of the MRN complex and ATR recruitment. A Schematic diagram of RNA pulldown and mass spectrometry to identify scaRNA2 binding proteins. B-C Representative images of silver staining of proteins after electrophoretic separation. Proteins including RBMX, Mre11, RPA, and replication factors such as RFC2-5 were identified. The number of peptides and coverage are indicated. D The binding of scaRNA2 with RPA2, Mre11 and RAD51 was determined with RIP experiments with a specific antibody in irradiated HCT116 cells. ***P < 0.001, **P < 0.01 compared with the IgG group. E An RNA pulldown assay was performed with biotin-labelled scaRNA2 and protein lysis from irradiated HCT116 cells. The MRN complex, RPA2 and Exo1 were detected using a western blot analysis. F The binding of scaRNA2 fragments with the MRN complex was determined with biotin-labelled scaRNA2 FL, scaRNA2 F1 and scaRNA2 F2 transcripts. G-H In sg-ctrl and scaRNA2 knockdown HCT116 cells, immunoprecipitation was conducted with ATR (G) or Mre11 (H) antibody to detect the binding between MRN members and ATR at 8 h after 8 Gy irradiation. I Schematic model of MRN assembly and ATR recruitment after irradiation in the presence and absence of scaRNA2