Importance of CNOT8 Deadenylase Subunit in DNA Damage Responses Following Ionizing Radiation (IR).

Background The Ccr4-Not protein complex (CNOT complex) is a key regulator of gene expression in eukaryotic cells. Ccr4-Not Complex is composed of at least nine conserved subunits in mammalian cells with two main enzymatic activities. CNOT8 is a subunit of the complex with deadenylase activity that interacts transiently with the CNOT6 or CNOT6L subunits. Here, we focused on the role of the human CNOT8 subunit in the DNA damage response (DDR). Methods Cell viability was assessed to measure ATP level using a Cell Titer-Glo Luminescence reagent up to 4 days' post CNOT8 siRNA transfection. In addition, expression level of phosphorylated proteins in signalling pathways were detected by western blotting and immunofluorescence microscopy. CNOT8- depleted Hela cells post- 3 Gy ionizing radiation (IR) treatment were considered as a control. Results Our results from cell viability assays indicated a significant reduction at 72-hour post CNOT8 siRNA transfection (p= 0.04). Western blot analysis showed slightly alteration in the phosphorylation of DNA damage response (DDR) proteins in CNOT8-depleted HeLa cells following treatment with ionizing radiation (IR). Increased foci formation of γH2AX, RPA, 53BP1, and RAD51 foci was observed after IR in CNOT8-depleted cells compared to the control cells. Conclusion We conclude that CNOT8 deadenylase subunit is involved in the cellular response to DNA damage.