The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent.
The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent.
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DOI：
10.1093/nar/gkv895
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发表时间：
2015-11-16
影响因子：
14.9
通讯作者：
Helm M
中科院分区：
文献类型：
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作者：
Hauenschild R;Tserovski L;Schmid K;Thüring K;Winz ML;Sharma S;Entian KD;Wacheul L;Lafontaine DL;Anderson J;Alfonzo J;Hildebrandt A;Jäschke A;Motorin Y;Helm M
逆转录(RT)和高通量测序的结合已经成为一种强大的组合，通过分析流产的RT产物或将错配的dNTPs掺入到cDNA中来检测RNA中的修饰核苷酸。在这里，我们同时就模板RNA中N-1-甲基腺苷(M1a)的出现详细分析这两个参数。这种自然发生的修饰与结构效应有关，但它也被认为是核糖体RNA中抗生素耐药性的媒介。在与二甲基硫酸酯的结构探测实验中，M1a通常通过RT-拦阻检测到。一种专门开发的RNA-Seq协议被量身定做，用于同时分析RT-阻止和误掺入模式。通过对各种天然和合成RNA制剂的应用，我们发现了M1A的一个特征特征，除了阻滞率外，它还具有误掺入的重要成分。详细的分析表明，签名取决于RNA结构和模板RNA中M1a的3‘核苷酸的性质，这意味着它是序列相关的。M1a的RT-Signature用于检查和确认可疑的修饰位点，并导致在锥虫tRNA中鉴定出迄今未知的M1a残基。
The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m1A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m1A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored to the simultaneous analysis of RT-arrest and misincorporation patterns. By application to a variety of native and synthetic RNA preparations, we found a characteristic signature of m1A, which, in addition to an arrest rate, features misincorporation as a significant component. Detailed analysis suggests that the signature depends on RNA structure and on the nature of the nucleotide 3′ of m1A in the template RNA, meaning it is sequence dependent. The RT-signature of m1A was used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m1A residues in trypanosomal tRNA.