Global and single-nucleotide resolution detection of 7-methylguanosine in RNA

ABSTRACT

RNA modifications, including N-7-methylguanosine (m⁷G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m⁷G modifications is of paramount significance, given recent associations between altered m⁷G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m⁷G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m⁷G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m⁷G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH₄, neutral pH and the addition of 7-methylguanosine monophosphate (m⁷GMP) during the reducing reaction. Notably, compared to NaBH₄-based methods, Bo-Seq enhances the m⁷G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m⁷G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m⁷G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m⁷G in gene expression and its implications in cancer biology.

KEYWORDS:

• 7-methylguanosine
• cleavage
• deep sequencing
• epitranscriptome
• RNA modification
• transcriptome-wide detection methods

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

SD’A and RG-V performed the methodology, analysed data and wrote the manuscript. PV performed primer extension assays. NM-C, LB, MG-L and AMA performed the methodology. SD and DK analysed sequencing data. AH supervised data analyses. SB conceptualized the project, got the funding, supervised, designed experiments, analysed the data and wrote the manuscript.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15476286.2024.2337493

Additional information

Funding

This work was funded by the Spanish Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”, by the “European Union” [SAF2016-78667-R, PID2019-111692RB-I00, and PID2022-139598OB-I00]. In addition, we acknowledge funding from The Scientific Foundation AECC [LABAE19040BLAN], and the University of Salamanca and Fundación Memoria de Samuel Solorzano Barruso [FS/21-2018, FS/25-2019, FS/36-2020] and Consejería de Educación de la Junta de Castilla y León [CSI264P20] to S.B. S.B. was supported by Ramón y Cajal program from Spanish Ministry of Economy and Innovation [MINECO] [RYC-2014-16550]. R.G-V. was supported by a predoctoral fellowship funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future” (BES-2017-080530). The Instituto de Biología Molecular y Celular del Cancer is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia co-financed by the Castilla–León autonomous government and the European Regional Development Fund [CLC–2017–01]. S.B., R.G-V., A.H. and D.K. belong to the Spanish National Research Council (CSIC)’s Cancer Hub.