An Investigation of RNA Methylations with Biophysical Approaches in a Cervical Cancer Cell Model
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...
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2024-11-01
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| author | Buket Sağlam Onur Akkuş Azime Akçaöz-Alasar Çağatay Ceylan Günnur Güler Bünyamin Akgül |
| author_facet | Buket Sağlam Onur Akkuş Azime Akçaöz-Alasar Çağatay Ceylan Günnur Güler Bünyamin Akgül |
| author_sort | Buket Sağlam |
| collection | DOAJ |
| description | 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 m<sup>6</sup>A-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 m<sup>6</sup>A 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 m<sup>1</sup>A and m<sup>6</sup>A 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. |
| format | Article |
| id | doaj-art-b641adbd93074604b52e649ab6dc8081 |
| institution | OA Journals |
| issn | 2073-4409 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | MDPI AG |
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| series | Cells |
| spelling | doaj-art-b641adbd93074604b52e649ab6dc80812025-08-20T01:53:49ZengMDPI AGCells2073-44092024-11-011322183210.3390/cells13221832An Investigation of RNA Methylations with Biophysical Approaches in a Cervical Cancer Cell ModelBuket Sağlam0Onur Akkuş1Azime Akçaöz-Alasar2Çağatay Ceylan3Günnur Güler4Bünyamin Akgül5Noncoding RNA Laboratory, Department of Molecular Biology and Genetics, İzmir Institute of Technology, 35430 Izmir, TürkiyeBiophysics Laboratory, Department of Physics, İzmir Institute of Technology, 35430 Izmir, TürkiyeNoncoding RNA Laboratory, Department of Molecular Biology and Genetics, İzmir Institute of Technology, 35430 Izmir, TürkiyeDepartment of Food Engineering, İzmir Institute of Technology, 35430 Izmir, TürkiyeBiophysics Laboratory, Department of Physics, İzmir Institute of Technology, 35430 Izmir, TürkiyeNoncoding RNA Laboratory, Department of Molecular Biology and Genetics, İzmir Institute of Technology, 35430 Izmir, TürkiyeRNA 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 m<sup>6</sup>A-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 m<sup>6</sup>A 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 m<sup>1</sup>A and m<sup>6</sup>A 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.https://www.mdpi.com/2073-4409/13/22/1832m<sup>6</sup>A<i>N</i><sup>6</sup>-methyladenosineFT-IRcircular dichroismapoptosisTNF-α |
| spellingShingle | Buket Sağlam Onur Akkuş Azime Akçaöz-Alasar Çağatay Ceylan Günnur Güler Bünyamin Akgül An Investigation of RNA Methylations with Biophysical Approaches in a Cervical Cancer Cell Model Cells m<sup>6</sup>A <i>N</i><sup>6</sup>-methyladenosine FT-IR circular dichroism apoptosis TNF-α |
| title | An Investigation of RNA Methylations with Biophysical Approaches in a Cervical Cancer Cell Model |
| title_full | An Investigation of RNA Methylations with Biophysical Approaches in a Cervical Cancer Cell Model |
| title_fullStr | An Investigation of RNA Methylations with Biophysical Approaches in a Cervical Cancer Cell Model |
| title_full_unstemmed | An Investigation of RNA Methylations with Biophysical Approaches in a Cervical Cancer Cell Model |
| title_short | An Investigation of RNA Methylations with Biophysical Approaches in a Cervical Cancer Cell Model |
| title_sort | investigation of rna methylations with biophysical approaches in a cervical cancer cell model |
| topic | m<sup>6</sup>A <i>N</i><sup>6</sup>-methyladenosine FT-IR circular dichroism apoptosis TNF-α |
| url | https://www.mdpi.com/2073-4409/13/22/1832 |
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