Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.
Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.
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DOI：
10.1371/journal.pone.0143756
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Abe T
中科院分区：
文献类型：
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作者：
Mishima E;Jinno D;Akiyama Y;Itoh K;Nankumo S;Shima H;Kikuchi K;Takeuchi Y;Elkordy A;Suzuki T;Niizuma K;Ito S;Tomioka Y;Abe T
RNA修饰的生物学作用在很大程度上仍不清楚。因此，开发一种检测RNA修饰的方法对于进一步澄清是重要的。我们开发了一种检测RNA修饰的方法，称为免疫Northern印迹(INB)分析，并在此介绍了它的各种功能。这种方法包括使用聚丙烯酰胺或琼脂糖凝胶电泳法分离RNA，然后转移到尼龙膜上，然后使用针对修饰核苷的抗体进行免疫印迹，以检测特定的修饰。我们证实，带有1-甲基腺苷(M1A)、N6-甲基腺苷(M6A)、假尿苷和5-甲基胞苷(M5C)抗体的INB在不同物种和细胞器的不同RNA中显示出不同的修饰。INB与抗M5C抗体的交叉反应表明，该抗体与DNA上的另一种修饰发生交叉反应，表明该方法可应用于修饰核苷抗体的表征。此外，将INB与真核tRNA中高度特异性的修饰M1a的抗体结合使用，我们检测到细胞应激反应下的tRNA衍生片段tiRNAs，这表明我们可以应用于追踪含有特定修饰的靶RNA。使用抗M6A抗体的INB证实了M6A被特定的去甲基酶、脂肪质量和肥胖相关蛋白(FTO)和ALKBH5去甲基化，这表明它应用于对分离的RNA中的靶修饰进行定量。此外，INB证明FTO和ALKBH5基因的敲除不仅增加了小RNA中m6A的修饰，也增加了m6A的修饰。该方法具有较高的特异性、灵敏度和定量能力，可在常规实验装置上使用。因此，该方法对RNA修饰和新陈代谢的研究具有重要意义。
The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.