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.

RNA methylation adds a second layer of genetic information that dictates the post-transcriptional fate of RNAs. Although various methods exist that enable the analysis of RNA methylation in a site-specific or transcriptome-wide manner, whether biophysical approaches can be employed to such analyses is unexplored. In this study, Fourier-transform infrared (FT-IR) and circular dichroism (CD) spectroscopy are employed to examine the methylation status of both synthetic and cellular RNAs. The results show that FT-IR spectroscopy is perfectly capable of quantitatively distinguishing synthetic m6A-methylated RNAs from un-methylated ones. Subsequently, FT-IR spectroscopy is successfully employed to assess the changes in the extent of total RNA methylation upon the knockdown of the m6A writer, METTL3, in HeLa cells. In addition, the same approach is shown to accurately detect reduction in total RNA methylation upon the treatment of HeLa cells with tumor necrosis factor alpha (TNF-α). It is also demonstrated that m1A and m6A methylation induce quite a distinct secondary structure on RNAs, as evident from CD spectra. These results strongly suggest that both FT-IR and CD spectroscopy methods can be exploited to uncover biophysical properties impinged on RNAs by methyl moieties, providing a fast, convenient and cheap alternative to the existing methods.

Background/aim: Cancer is a complex disease that involves both genetic and epigenetic factors. While emerging evidence clearly suggests that changes in epitranscriptomics play a crucial role in cancer pathogenesis, a comprehensive understanding of the writers, erasers, and readers of epitranscriptomic processes, particularly under apoptotic conditions remains lacking. The aim of this study was to uncover the changes in the expression of m6A RNA modifiers under apoptotic conditions across various cancer cell lines. Materials and methods: Initially, we quantified the abundance of m6A RNA modifiers in cervix (HeLa and ME180), breast (MCF7 and MDA-MB-231), lung (A549 and H1299), and colon (CaCo2 and HCT116) cancer cell lines using qPCR. Subsequently, we induced apoptosis using cisplatin and tumor necrosis factor-alpha (TNF-α) to activate intrinsic and extrinsic pathways, respectively, and assessed apoptosis rates via flow cytometry. Further, we examined the transcript abundance of m6A RNA modifiers under apoptotic conditions in cervix, breast, and lung cancer cell lines using qPCR. Results: Overall, treatment with cisplatin increased the abundance of m6A modifiers, whereas TNF-α treatment decreased their expression in cervix, breast, and lung cancer cell lines. Specifically, cisplatin-induced apoptosis, but not TNF-α-mediated apoptosis, resulted in decreased abundance of METTL14 and FTO transcripts. Additionally, cisplatin treatment drastically reduced the abundance of IGF2BP2 and IGF2BP3 readers. Conclusion: These results suggest that the differential response of cancer cells to apoptotic inducers may be partially attributed to the expression of m6A RNA modifiers.

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 m ⁶ A marks is unknown. We employed a genomewide approach to examine the extent of m ⁶ A RNA modifications under TNF-α-modulated apoptotic conditions in HeLa cells. miCLIP-seq analyses revealed a plethora of m ⁶ A marks on 632 target mRNAs with an enrichment on 99 mRNAs associated with apoptosis. Interestingly, the m ⁶ A 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 m ⁶ A methylation marks exist in cells and TNF-α-METTL3-TP53INP1 axis modulates TNF-α-mediated apoptosis in HeLa cells.

Nucleic acid methylation is a fundamental epigenetic regulation that influences gene expression and is increasingly explored as a biomarker in cancer biology. However, conventional methods employed for methylation analysis are often labor-intensive, require comprehensive sample preparation and lack real-time structural sensitivity. Herein, we report experimentally validated spectroscopic framework for a quantitative label-free detection of methyl group (-CH₃) signatures and structural features of nucleic acids by using attenuated total reflection Fourier transform infrared (ATR-FTIR) and circular dichroism (CD) spectroscopic techniques. Accordingly, concentration-dependent detection thresholds and spectral resolution limits for DNA and RNA methylation were determined. ATR-FTIR data revealed distinct IR peaks attributed to the asymmetric and symmetric stretching and bending vibrations of the CH₃ groups, and displayed improved spectral separation appearing at intermediate concentrations (~500 ng/µL) of nucleic acids. Comparison of methylated RNA oligonucleotides and cell-derived RNA samples confirmed the robustness of methyl-sensitive spectral markers. CD spectroscopy demonstrated a strong linear correlation between DNA concentration and ellipticity at 277 nm, reflecting the excitonic coupling theory. RNA gave rise to detectable CD signals at lower concentrations (~25 ng/µL) while DNA required around 100 ng/µL for reliable structural interpretation, reflecting intrinsic differences between A-form and B-form helices. Our study provides (i) experimentally determined lower detection limits, (ii) validated IR vibrational fingerprints of methylation-associated groups and (iii) a combined spectroscopic analysis suitable for rapid and cost-effective examination of nucleic acid methylation. Thus, the current study lays the groundwork for future applications targeting site-specific modifications such as m⁶A and 5-methylcytosine in biomolecules.