Global and single-nucleotide resolution detection of 7-methylguanosine in RNA
Silvia D’Ambrosi,
Raquel García-Vílchez,
Darek Kedra,
Patrice Vitali,
Nuria Macias-Cámara,
Laura Bárcena,
Monika Gonzalez-Lopez,
Ana M. Aransay,
Sabine Dietmann,
Antonio Hurtado,
Sandra Blanco
Affiliations
Silvia D’Ambrosi
Department of Neurosurgery, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
Raquel García-Vílchez
Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-University of Salamanca, Salamanca, Spain
Darek Kedra
Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-University of Salamanca, Salamanca, Spain
Patrice Vitali
Molecular, Cellular and Developmental Biology unit (MCD), Centre de Biologie Integrative (CBI), University of Toulouse, UPS, CNRS, Toulouse, France
Nuria Macias-Cámara
CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain
Laura Bárcena
CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain
Monika Gonzalez-Lopez
CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain
Ana M. Aransay
CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain
Sabine Dietmann
Department of Developmental Biology, Washington University School of Medicine in St. Louis, St. Louis, MO, USA
Antonio Hurtado
Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-University of Salamanca, Salamanca, Spain
Sandra Blanco
CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain
ABSTRACTRNA 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.
