Epitranscriptomics m⁶A analyses reveal distinct m⁶A marks under tumor necrosis factor α (TNF-α)-induced apoptotic...

Tumor necrosis factor-α (TNF-α) is a ligand that induces both intrinsic and extrinsic apoptotic pathways in HeLa cells by modulating complex gene regulatory mechanisms. However, the full spectrum of TNF-α-modulated epitranscriptomic m⁶A marks is unknown. We employed a genomewide approach to examine the extent of m⁶A RNA modifications under TNF-α-modulated apoptotic conditions in HeLa cells. miCLIP-seq analyses revealed a plethora of m⁶A marks on 632 target mRNAs with an enrichment on 99 mRNAs associated with apoptosis. Interestingly, the m⁶A RNA modification patterns were quite different under cisplatin- and TNF-α-mediated apoptotic conditions. We then examined the abundance and translational efficiencies of several mRNAs under METTL3 knockdown and/or TNF-α treatment conditions. Our analyses showed changes in the translational efficiency of TP53INP1 mRNA based on the polysome profile analyses. Additionally, TP53INP1 protein amount was modulated by METTL3 knockdown upon TNF-α treatment but not CP treatment, suggesting the existence of a pathway-specific METTL3-TP53INP1 axis. Congruently, METLL3 knockdown sensitized HeLa cells to TNF-α-mediated apoptosis, which was also validated in a zebrafish larval xenograft model. These results suggest that apoptotic pathway-specific m⁶A methylation marks exist in cells and TNF-α-METTL3-TP53INP1 axis modulates TNF-α-mediated apoptosis in HeLa cells.