Single-base resolution mapping of 2'-O-methylation sites by an exoribonuclease-enriched chemical method
Single-base resolution mapping of 2'-O-methylation sites by an exoribonuclease-enriched chemical method
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通过富含外切核糖核酸酶的化学方法对 2-O-甲基化位点进行单碱基分辨率作图
DOI：
10.1007/s11427-022-2210-0
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通讯作者：
Jianhua Yang
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作者：
Ping Zhang;Junhong Huang;Wujian Zheng;Lifan Chen;Shurong Liu;Anrui Liu;Jiayi Ye;Jie Zhou;Zhirong Chen;Qiaojuan Huang;Shun Liu;Keren Zhou;Lianghu Qu;Bin Li;Jianhua Yang
2 '-O-甲基化（Nm）是RNA表观遗传修饰中最丰富的一种，在基因表达的转录后调控中起着重要作用。目前的Nm作图方法通常限于高度丰富的RNA，并且在mRNA或相对罕见的非编码RNA（ncRNA）中具有显著的技术障碍。在这里，我们开发了一种新的方法，通过使用RNA核糖核酸外切酶和高碘酸盐氧化反应性来消除2 '-羟基化（2'-OH）核苷，结合测序（Nm-REP-seq）来富集Nm位点。我们揭示了几种新的类含Nm的ncRNA以及mRNA在人类，小鼠和果蝇。我们发现一些新的Nm位点存在于不同的tRNA的固定位置，并且是由snoRNA介导的纤维素（FBL）甲基转移酶的潜在底物。重要的是，我们首次发现Nm位于各种类型的ncRNA及其衍生片段的3 '端。我们的方法精确地重新定义了Nm的全基因组分布，并为Nm介导的基因调控的功能研究提供了新的技术。
2'-O-methylation (Nm) is one of the most abundant RNA epigenetic modifications and plays a vital role in the post-transcriptional regulation of gene expression. Current Nm mapping approaches are normally limited to highly abundant RNAs and have significant technical hurdles in mRNAs or relatively rare non-coding RNAs (ncRNAs). Here, we developed a new method for enriching Nm sites by using RNA exoribonuclease and periodate oxidation reactivity to eliminate 2'-hydroxylated (2'-OH) nucleosides, coupled with sequencing (Nm-REP-seq). We revealed several novel classes of Nm-containing ncRNAs as well as mRNAs in humans, mice, and drosophila. We found that some novel Nm sites are present at fixed positions in different tRNAs and are potential substrates of fibrillarin (FBL) methyltransferase mediated by snoRNAs. Importantly, we discovered, for the first time, that Nm located at the 3'-end of various types of ncRNAs and fragments derived from them. Our approach precisely redefines the genome-wide distribution of Nm and provides new technologies for functional studies of Nm-mediated gene regulation.