High-throughput single-base resolution mapping of RNA 2΄-O-methylated residues.
High-throughput single-base resolution mapping of RNA 2΄-O-methylated residues.
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DOI：
10.1093/nar/gkw810
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发表时间：
2017-02-17
影响因子：
14.9
通讯作者：
Oliviero S
中科院分区：
文献类型：
--
作者：
Incarnato D;Anselmi F;Morandi E;Neri F;Maldotti M;Rapelli S;Parlato C;Basile G;Oliviero S
转录组的功能表征需要系统研究RNA转录后修饰的工具。核糖部分的2 <$-O-methylation（2 <$-OMe）是RNA最丰富的转录后修饰之一，但由于缺乏可靠的高通量作图方法，其系统分析比较困难。我们在这里描述了一种新的高通量方法，命名为2 OMe-seq，它能够快速和准确的映射在单碱基分辨率，和相对定量，2个OMe-OMe修饰的残基。我们将我们的方法与其他最先进的方法进行比较，并表明它具有更高的灵敏度和特异性。通过将2 OMe-seq应用于HeLa细胞，我们表明它能够恢复核糖体RNA上大部分注释的2个OMe位点。通过在小鼠胚胎干细胞（ESC）中进行原纤蛋白甲基转移酶的敲低，我们显示了2 OMe-seq捕获2 β-O-甲基化水平变化的能力。此外，使用2 OMe-seq数据，我们在这里报告了12个先前未注释的跨18 S和28 S rRNA的2-OMe位点的发现，其中11个在人类和小鼠细胞中都是保守的，并为所有位点分配了相应的snoRNA。我们的方法扩展了用于RNA转录后修饰的全转录组映射的方法，并有望为这种修饰的作用提供新的见解。
Functional characterization of the transcriptome requires tools for the systematic investigation of RNA post-transcriptional modifications. 2΄-O-methylation (2΄-OMe) of the ribose moiety is one of the most abundant post-transcriptional modifications of RNA, although its systematic analysis is difficult due to the lack of reliable high-throughput mapping methods. We describe here a novel high-throughput approach, named 2OMe-seq, that enables fast and accurate mapping at single-base resolution, and relative quantitation, of 2΄-OMe modified residues. We compare our method to other state-of-art approaches, and show that it achieves higher sensitivity and specificity. By applying 2OMe-seq to HeLa cells, we show that it is able to recover the majority of the annotated 2΄-OMe sites on ribosomal RNA. By performing knockdown of the Fibrillarin methyltransferase in mouse embryonic stem cells (ESCs) we show the ability of 2OMe-seq to capture 2΄-O-Methylation level variations. Moreover, using 2OMe-seq data we here report the discovery of 12 previously unannotated 2΄-OMe sites across 18S and 28S rRNAs, 11 of which are conserved in both human and mouse cells, and assigned the respective snoRNAs for all sites. Our approach expands the repertoire of methods for transcriptome-wide mapping of RNA post-transcriptional modifications, and promises to provide novel insights into the role of this modification.