Arango Daniel, Sturgill David, Oberdoerffer Shalini
Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Bio Protoc. 2019 Jun 20;9(12):e3278. doi: 10.21769/BioProtoc.3278.
Generation of the epitranscriptome through chemical modifications of protein-coding messenger RNAs (mRNAs) has emerged as a new mechanism of post-transcriptional gene regulation. While most mRNA modifications are methylation events, a single acetylated ribonucleoside has been described in eukaryotes, occurring at the N4-position of cytidine (N4-acetylcytidine or ac4C). Using a combination of antibody-based enrichment of acetylated regions and deep sequencing, we recently reported ac4C as a novel mRNA modification that is catalyzed by the N-acetyltransferase enzyme NAT10. In this protocol, we describe in detail the procedures to identify acetylated mRNA regions transcriptome-wide using acetylated RNA immunoprecipitation and sequencing (acRIP-seq).
通过对蛋白质编码信使核糖核酸（mRNA）进行化学修饰来生成表观转录组，已成为一种新的转录后基因调控机制。虽然大多数mRNA修饰是甲基化事件，但在真核生物中已发现一种单一的乙酰化核糖核苷，它出现在胞苷的N4位（N4-乙酰胞苷或ac4C）。我们最近结合基于抗体的乙酰化区域富集和深度测序，报告了ac4C是一种由N-乙酰转移酶NAT10催化的新型mRNA修饰。在本方案中，我们详细描述了使用乙酰化RNA免疫沉淀和测序（acRIP-seq）在全转录组范围内鉴定乙酰化mRNA区域的程序。
