Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, USA.
Methods Mol Biol. 2024;2832:47-55. doi: 10.1007/978-1-0716-3973-3_3.
Recent advancements in detection and mapping methods have enabled researchers to uncover the biological importance of RNA chemical modifications, which play a vital role in post-transcriptional gene regulation. Although numerous types of RNA modifications have been identified in higher eukaryotes, only a few have been extensively studied for their biological functions. Of these, N-methyladenosine (mA) is the most prevalent and important mRNA modification that influences various aspects of RNA metabolism, including mRNA stability, degradation, splicing, alternative polyadenylation, export, and localization, as well as translation. Thus, they have implications for a variety of biological processes, including growth, development, and stress responses. The mA deposition or removal on transcripts is dynamic and is altered in response to internal and external cues. Because this mark can alter gene expression under stress conditions, it is essential to identify the transcripts that can acquire or lose this epitranscriptomic mark upon exposure to stress conditions. Here we describe a step-by-step protocol for identifying stress-responsive transcriptome-wide mA changes using RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq).
最近在检测和绘图方法方面的进展使研究人员能够揭示 RNA 化学修饰的生物学重要性，这些修饰在转录后基因调控中起着至关重要的作用。尽管在高等真核生物中已经鉴定出了许多类型的 RNA 修饰，但只有少数几种修饰因其生物学功能而得到了广泛研究。其中，N6-甲基腺苷（m6A）是最普遍和最重要的 mRNA 修饰，它影响 RNA 代谢的各个方面，包括 mRNA 稳定性、降解、剪接、可变多聚腺苷酸化、输出和定位以及翻译。因此，它们对包括生长、发育和应激反应在内的各种生物学过程都有影响。转录本上的 m6A 沉积或去除是动态的，并响应内部和外部线索而改变。由于这种标记可以在应激条件下改变基因表达，因此必须识别在暴露于应激条件下可以获得或失去这种转录后修饰标记的转录本。在这里，我们描述了一种使用 RNA 免疫沉淀 followed by 高通量测序（MeRIP-seq）来鉴定应激响应的全转录组 m6A 变化的逐步方案。
