Although their functional importance in human biology has not been extensively studied, it has recently been partially revealed that some modifications modulate a variety of RNA functions and biological processes, and they are linked to various human diseases. RNAs contain a wide variety of post-transcriptional nucleoside modifications, which vary greatly between different RNA molecules, organisms and organelles. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedĭata Availability: All relevant data are within the paper and Supporting Information files.įunding: This study was funded by JSPS KAKENHI Grant Number 26860624 to EM, JSPS KAKENHI Grant Number 23390033 to TA, and JSPS KAKENHI Grant Number 26305007 to TA.Ĭompeting interests: The authors have declared that no competing interests exist. Received: AugAccepted: NovemPublished: November 25, 2015Ĭopyright: © 2015 Mishima et al. PLoS ONE 10(11):Įditor: Thomas Preiss, The John Curtin School of Medical Research, AUSTRALIA (2015) Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides. Therefore, this method would be useful for research on RNA modifications and metabolism.Ĭitation: Mishima E, Jinno D, Akiyama Y, Itoh K, Nankumo S, Shima H, et al. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m 6A modification in small RNAs as well as in mRNA. INB with the anti-m 6A antibody confirmed the demethylation of m 6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Additionally, using INB with the antibody for m 1A, 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-m 5C 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. We confirmed that INB with the antibodies for 1-methyladenosine (m 1A), N6-methyladenosine (m 6A), pseudouridine, and 5-methylcytidine (m 5C) showed different modifications in a variety of RNAs from various species and organelles. 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 developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. Thus, developing a method for detecting RNA modifications is important for further clarification. J Vis Exp 118:54672.The biological roles of RNA modifications are still largely not understood. Wang CY, Lin MH, Su HT (2016) A method for measuring RNA N 6-methyladenosine modifications in cells and tissues.Feederle R, Schepers A (2017) Antibodies specific for nucleic acid modifications.Yamasaki S, Ivanov P, Hu GF, Anderson P (2009) Angiogenin cleaves tRNA and promotes stress-induced translational repression.Mishima E, Inoue C, Saigusa D, Inoue R, Ito K, Suzuki Y, Jinno D, Tsukui Y, Akamatsu Y, Araki M, Araki K, Shimizu R, Shinke H, Suzuki T, Takeuchi Y, Shima H, Akiyama Y, Toyohara T, Suzuki C, Saiki Y, Tominaga T, Miyagi S, Kawagisihi N, Soga T, Ohkubo T, Yamamura K, Imai Y, Masuda S, Sabbisetti V, Ichimura T, Mount DB, Bonventre JV, Ito S, Tomioka Y, Itoh K, Abe T (2014) Conformational change in transfer RNA is an early indicator of acute cellular damage.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 (2015) Immuno-northern blotting: detection of RNA modifications by using antibodies against modified nucleosides.
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