Detecting and Mapping N6-Methyladenosine on RNA/DNA Hybrids.
Detecting and Mapping N6-Methyladenosine on RNA/DNA Hybrids.
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检测和绘制 RNA/DNA 杂交体上的 N6-甲基腺苷。
DOI：
10.1007/978-1-0716-2477-7_22
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Abakir A
中科院分区：
文献类型：
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作者：
Abakir A
N6-甲基腺苷（m6 A）是真核生物中基因表达转录后调控所必需的RNA修饰。我们最近证明，m6 A装饰的RNA组件的R-环，特定的核酸结构组成的RNA/DNA杂交和单链的非模板DNA，这是一个主要来源的遗传不稳定性，并在同一时间，有助于调节基因表达在哺乳动物细胞。根据越来越多的实验证据，腺苷甲基化影响这些结构的稳定性，并可能影响其代谢的各个方面。在这里，我们提出了两种方法检测和分析的m6 A-含有RNA/DNA杂交：一个免疫染色协议，允许调查其在真核细胞和m6 A-DNA免疫沉淀（DIP），抗体为基础的技术，允许其基因组定位和位点特异性分析的空间分布。除了以m6 A为重点的研究外，这些方法也有助于阐明其他RNA修饰在R环生物学中的功能作用。
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.