Genomewide m6A Mapping Uncovers Dynamic Changes in the m6A Epitranscriptome of Cisplatin-Treated Apoptotic HeLa Cells

Abstract

Cisplatin (CP), which is a conventional cancer chemotherapeutic drug, induces apoptosis by modulating a diverse array of gene regulatory mechanisms. However, cisplatin-mediated changes in the m6A methylome are unknown. We employed an m6A miCLIP-seq approach to investigate the effect of m6A methylation marks under cisplatin-mediated apoptotic conditions on HeLa cells. Our high-resolution approach revealed numerous m6A marks on 972 target mRNAs with an enrichment on 132 apoptotic mRNAs. We tracked the fate of differentially methylated candidate mRNAs under METTL3 knockdown and cisplatin treatment conditions. Polysome profile analyses revealed perturbations in the translational efficiency of PMAIP1 and PHLDA1 transcripts. Congruently, PMAIP1 amounts were dependent on METTL3. Additionally, cisplatin-mediated apoptosis was sensitized by METTL3 knockdown. These results suggest that apoptotic pathways are modulated by m6A methylation events and that the METTL3–PMAIP1 axis modulates cisplatin-mediated apoptosis in HeLa cells.

Epitranscriptomics m6A analyses reveal distinct m6A marks under tumor necrosis factor α (TNF‐α)‐induced apoptotic conditions in HeLa cells

“…Our analyses revealed that METTL3 knockdown perturbs the polysomal association of PAWR, IFI6, and TP53INP1, the nonpolysomal (mRNP or monosome) association of IFI6, GADD45B, HRK, BMF, and BCL2A1, without any major effects on the translation efficiency of PHLDA1 and CARD14 (Figure S2). To interrogate the polysomal association of the apoptotic candidate transcripts in the presence or absence of TNF-α in METTL3 knockdown cells, we performed polysome profiling analyses with HeLa cells as previously reported (Alasar et al, 2022). TNF-α treatment did not change the polysome profile of METTL3 knockdown cells compared to the control CHX-treated cells (Figure 4a).…”

“…qPCR and western blot analyses revealed changes in the expression of writers (Figure 1). Similarly, treatment of HeLa cells with CP downregulates the expression of METTL3 and METTL14 (Alasar et al, 2022), suggesting that downregulation of writer proteins may be a common feature in apoptotic cells independent of the inducers.…”

“…It is very interesting that METTL3 and METTL14 protein amounts are downregulated upon both CP and TNF-α treatments ( [Alasar et al, 2022]; Figure 1c). There appears to be a consistent reduction in the writer protein amounts irrespective of the apoptotic inducer.…”

“…The METTL3 overexpression plasmid pcDNA3.1(+)-METTL3 was described previously (Alasar et al, 2022). HeLa cells were transfected with pcDNA3.1(+)-METTL3 or pcDNA3.1(+) using Fugene HD transfection reagent (Promega) according to the manufacturer's protocol.…”

YTHDF1 Promotes Bladder Cancer Cell Proliferation via the METTL3/YTHDF1–RPN2–PI3K/AKT/mTOR Axis

“…YTHDF1 can affect the progression of ovarian, breast and small cell lung cancers, and it can drive hypoxia-induced autophagy in hepatocellular carcinoma, but no relevant studies have been reported on BLCA [ 15 , 16 , 17 , 18 ]. Decreased METTL3 expression increased the sensitivity of Hela cells to cisplatin, and increased METTL3 expression in bladder cancer cells increased the resistance of bladder cancer cells to cisplatin [ 12 , 19 ]. In m6A modification, the writer plays different roles by binding to different readers, so we explored the effects of the writer METTL3 and the reader YTHDF1 on BLCA cell proliferation and cisplatin sensitivity [ 14 , 20 , 21 , 22 , 23 ].…”