Abstract
Increasing evidence demonstrates that RNA nucleotides undergo epigenetic modifications, such as methylation on cytosine. Although the presence of modified bases on mRNA has been proven, their molecular significance is largely undefined. We describe here a methodology to dissect the timing of modification of cytosine to 5-methylcytosine (5mC or m5C) in relation to RNA elongation and processing. To do this we use chlorouridine and iodouridine, two synthetically modified nucleotide bases which can be recognized by RNA polymerase II and incorporated into nascent RNA. These modified bases are added to a cell culture for defined intervals of time, and then immunocytochemical staining using antibodies against the modified nucleotides is carried out. This procedure allows us to identify the range of time in which 5mC is produced in nascent mRNA. This method provides the ultra-resolution of electron microscopy and allows following nascent RNA molecules during their elongation.
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Acknowledgments
The authors would like to thank Ms. Francine Flach and Ms. Gloria Milanesi for excellent technical skill in preparing the ultrathin sections. This research was supported by the Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (2018–2022)—Dept. of Biology and Biotechnology “L. Spallanzani,” University of Pavia (to M.B.).
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Zannino, L., Siciliani, S., Biggiogera, M. (2020). Timing of Cytosine Methylation on Newly Synthesized RNA by Electron Microscopy. In: Hancock, R. (eds) The Nucleus . Methods in Molecular Biology, vol 2175. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0763-3_14
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DOI: https://doi.org/10.1007/978-1-0716-0763-3_14
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