Abstract
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.
Highlights
RNAs contain a wide variety of post-transcriptional nucleoside modifications [1, 2], which vary greatly between different RNA molecules, organisms and organelles
The specific bands are detected by subsequent incubation with the secondary antibody and the chemiluminescent reaction. The protocol of this method is similar to that of western blotting. We performed this immuno-northern blotting (INB) for the detection of the modified nucleosides with the antibodies against 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine (C), and 5-methylcytidine (m5C) in total RNAs isolated from various samples of mammalian cells, yeast (S. cerevisiae of strain BY4742 and W303), and bacteria (E. coli of strain DH5α and HST04) (Fig 2A and 2B)
Because the presence of m6A modifications was reported in eukaryotic mRNA, 18S rRNA, 28S rRNA, and small nuclear RNAs (snRNA) [12,13,14], the positive bands by the anti-m6A antibody were probably derived from these m6A-containing RNAs
Summary
RNAs contain a wide variety of post-transcriptional nucleoside modifications [1, 2], which vary greatly between different RNA molecules, organisms and organelles. 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 [3, 4]. Pseudouridine is the most abundant modified nucleoside in a wide variety of cellular RNAs containing tRNA, rRNA and small nuclear RNAs (snRNA), and it has been reported to contribute to the RNA-mediated cellular processes [4, 5]. To further elucidate the biological functions of RNA modifications, the development of analytical methods for detecting RNA modifications and their alterations is needed
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