Epigenetic Programme, Babraham Institute, Cambridge, UK.
General Department of Criminal Evidence, Ministry of Interior Affairs, Al-Dajeej, Kuwait.
Methods Mol Biol. 2022;2528:329-344. doi: 10.1007/978-1-0716-2477-7_22.
N6-methyladenosine (mA) is an RNA modification essential for posttranscriptional regulation of gene expression in eukaryotes. We recently demonstrated that mA 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 mA-containing RNA/DNA hybrids: an immunostaining protocol allowing investigation of their spatial distribution in eukaryotic cells and mA-DNA immunoprecipitation (DIP), an antibody-based technique that permits their genome mapping and locus-specific analysis. In addition to the mA-focused studies, these methodologies can also contribute to elucidating the functional roles of other RNA modifications in R-loop biology.
N6-甲基腺苷（mA）是真核生物中转录后基因表达调控所必需的 RNA 修饰。我们最近的研究表明，mA 修饰 R 环的 RNA 成分，R 环是由 RNA/DNA 杂交体和单链非模板 DNA 组成的特定核酸结构，它是遗传不稳定性的主要来源，同时也有助于哺乳动物细胞中基因表达的调控。根据越来越多的实验证据，腺苷甲基化会影响这些结构的稳定性，并可能影响它们代谢的各个方面。在这里，我们介绍了两种检测和分析含有 mA 的 RNA/DNA 杂交体的方法：一种免疫染色方案，可用于研究真核细胞中它们的空间分布，以及 mA-DNA 免疫沉淀（DIP），这是一种基于抗体的技术，可用于它们的基因组图谱绘制和特定基因座的分析。除了 mA 相关研究，这些方法还可以有助于阐明 R 环生物学中其他 RNA 修饰的功能作用。
