RNA binding protein NKAP protects glioblastoma cells from ferroptosis by promoting SLC7A11 mRNA splicing in an m6A-dependent manner

Ferroptosis is a form of cell death characterized by lipid peroxidation. Previous studies have reported that knockout of NF-κB activating protein (NKAP), an RNA-binding protein, increased lipid peroxidation level in naive T cells and induced cell death in colon Cancer cells. However, there was no literature reported the relationship between NKAP and Ferroptosis in glioblastoma cells. Notably, the mechanism of NKAP modulating Ferroptosis is still unknown. Here, we found NKAP knockdown induced cell death in glioblastoma cells. Silencing NKAP increased the cell sensitivity to Ferroptosis inducers both in vitro and in vivo. Exogenous overexpression of NKAP promoted cell resistance to Ferroptosis inducers by positively regulating a Ferroptosis defense protein, namely cystine/glutamate antiporter (SLC7A11). The regulation of SLC7A11 by NKAP can be weakened by the m⁶A methylation inhibitor cycloleucine and knockdown of the m⁶A writer METTL3. NKAP combined the "RGAC" motif which was exactly in line with the m⁶A motif "RGACH" (R = A/G, H = A/U/C) uncovered by the m⁶A-sequence. RNA Immunoprecipitation (RIP) and Co-Immunoprecipitation (Co-IP) proved the interaction between NKAP and m⁶A on SLC7A11 transcript. Following its binding to m⁶A, NKAP recruited the splicing factor proline and glutamine-rich (SFPQ) to recognize the splice site and then conducted transcription termination site (TTS) splicing event on SLC7A11 transcript and the retention of the last exon, screened by RNA-sequence and Mass Spectrometry (MS). In conclusion, NKAP acted as a new Ferroptosis suppressor by binding to m⁶A and then promoting SLC7A11 mRNA splicing and maturation.