Detecting and Mapping N6-Methyladenosine on RNA/DNA Hybrids. 2022

Abdulkadir Abakir, and Fahad Alenezi, and Alexey Ruzov
Epigenetic Programme, Babraham Institute, Cambridge, UK. abdulkadir.abakir@babraham.ac.uk.

N6-methyladenosine (m6A) is an RNA modification essential for posttranscriptional regulation of gene expression in eukaryotes. We recently demonstrated that m6A decorates the RNA components of R-loops, specific nucleic acid structures consisting of an RNA/DNA hybrid and a single strand of non-template DNA, that represent a major source of genetic instability and, at the same time, contribute to regulation of gene expression in mammalian cells. According to growing body of experimental evidence, adenosine methylation affects stability of these structures and potentially influences various aspects of their metabolism. Here, we present two methods for detection and analysis of m6A-containing RNA/DNA hybrids: an immunostaining protocol allowing investigation of their spatial distribution in eukaryotic cells and m6A-DNA immunoprecipitation (DIP), an antibody-based technique that permits their genome mapping and locus-specific analysis. In addition to the m6A-focused studies, these methodologies can also contribute to elucidating the functional roles of other RNA modifications in R-loop biology.

UI MeSH Term Description Entries
D008322 Mammals Warm-blooded vertebrate animals belonging to the class Mammalia, including all that possess hair and suckle their young. Mammalia,Mammal
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D000241 Adenosine A nucleoside that is composed of ADENINE and D-RIBOSE. Adenosine or adenosine derivatives play many important biological roles in addition to being components of DNA and RNA. Adenosine itself is a neurotransmitter. Adenocard,Adenoscan
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D012313 RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. (Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed) RNA, Non-Polyadenylated,Ribonucleic Acid,Gene Products, RNA,Non-Polyadenylated RNA,Acid, Ribonucleic,Non Polyadenylated RNA,RNA Gene Products,RNA, Non Polyadenylated
D047468 Immunoprecipitation The aggregation of soluble ANTIGENS with ANTIBODIES, alone or with antibody binding factors such as ANTI-ANTIBODIES or STAPHYLOCOCCAL PROTEIN A, into complexes large enough to fall out of solution. Co-Immunoprecipitation,Immune Precipitation,Co Immunoprecipitation,Co-Immunoprecipitations,Immune Precipitations,Precipitation, Immune,Precipitations, Immune

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