Noncoding RNA Laboratory, Department of Molecular Biology and Genetics, İzmir Institute of Technology, 35430 Izmir, Türkiye.
Biophysics Laboratory, Department of Physics, İzmir Institute of Technology, 35430 Izmir, Türkiye.
Cells. 2024 Nov 6;13(22):1832. doi: 10.3390/cells13221832.
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 mA-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 mA 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 mA and mA 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.
RNA 甲基化增加了第二层遗传信息，决定了 RNA 的转录后命运。尽管存在各种方法可以特异性或全转录组方式分析 RNA 甲基化，但生物物理方法是否可以用于此类分析尚待探索。在这项研究中，傅里叶变换红外（FT-IR）和圆二色性（CD）光谱用于检查合成和细胞 RNA 的甲基化状态。结果表明，FT-IR 光谱完全能够定量区分合成的 mA-甲基化 RNA 和未甲基化的 RNA。随后，FT-IR 光谱成功用于评估在 HeLa 细胞中敲低 mA 书写器 METTL3 后总 RNA 甲基化程度的变化。此外，还表明相同的方法可以准确检测到 TNF-α 处理 HeLa 细胞后总 RNA 甲基化的减少。还证明，mA 和 mA 甲基化会导致 RNA 上产生相当明显的二级结构，从 CD 光谱中可以明显看出。这些结果强烈表明，FT-IR 和 CD 光谱方法都可以用于揭示甲基基团对 RNA 的物理性质的影响，为现有方法提供了一种快速、方便和廉价的替代方法。
