ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments.
ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments.
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DOI：
10.1038/nmeth.3508
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发表时间：
2015-09
期刊：
影响因子：
48
通讯作者：
Lowe TM
中科院分区：
文献类型：
--
作者：
Cozen AE;Quartley E;Holmes AD;Hrabeta-Robinson E;Phizicky EM;Lowe TM
高通量RNA测序加速了对小RNA复杂调控作用的发现，但当这些修饰在RNA-seq文库制备过程中干扰逆转录时，含有修饰核苷的RNA可能会逃脱检测。在这里，我们描述了AlkB促进的RNA甲基化测序（ARM-Seq），它使用大肠杆菌AlkB进行预处理，以使1-甲基腺苷，3-甲基胞苷和1-甲基鸟苷脱甲基，所有这些都常见于转移RNA中。使用ARM-Seq的比较甲基化分析提供了这些修饰的第一个详细的转录组规模的图谱，并揭示了大量以前未检测到的、来自tRNA的甲基化小RNA。ARM-Seq证明，tRNA衍生的小RNA准确地重现了充分表征的酵母tRNA的m1A修饰状态，并对大量人类tRNA（包括tRNA前体和线粒体tRNA）产生了新的预测。因此，ARM-Seq为识别以前被忽视的甲基修饰的RNA提供了广泛的实用性，可以有效地监测甲基化状态，并可能揭示tRNA衍生的RNA作为生物标志物或信号分子的新作用。
High throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA Methylation sequencing (ARM-Seq) which uses pre-treatment with Escherichia coli AlkB to demethylate 1-methyladenosine, 3-methylcytidine, and 1-methylguanosine, all commonly found in transfer RNAs. Comparative methylation analysis using ARM-Seq provides the first detailed, transcriptome-scale map of these modifications, and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-Seq demonstrates that tRNA-derived small RNAs accurately recapitulate the m1A modification state for well-characterized yeast tRNAs, and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-Seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state, and may reveal new roles for tRNA-derived RNAs as biomarkers or signaling molecules.