Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides

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.

Genome-Wide Maps of m6A circRNAs Identify Widespread and Cell-Type-Specific Methylation Patterns that Are Distinct from mRNAs

“…The input RNA (rRNA-depleted, RNAse R-treated) and eluate fraction (m 6 A-positive fraction following m 6 A RNA immunoprecipitation (RIP)) both contain m 6 A modifications, while the supernatant (m 6 A-negative fraction) shows no evidence of m 6 A (Figure 1A bottom ). It is unlikely that the positive signal in the eluate fraction is due to tRNAs, which are also resistant to RNase R, because tRNAs are not modified by m 6 A in mammalian cells (Mishima et al, 2015). This conclusion is supported by the loss of the RNA peak around 75 nucleotides (nt) following m 6 A RIP (Figure S1A, eluate), which contains tRNAs (Holley et al, 1965).…”

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

“…This technique has also been used in conjunction with APM-containing polyacrylamide gels to identify the occurrence of thiolation on certain tRNA species [133,134,135,136,137]. Recently, a variation of the standard method, immuno-Northern blot, was provided which uses antibodies that specifically bind with modified nucleosides such as 1-methyladenosine (m 1 a), N 6 -methyladenosine (m 6 A), pseudouridine, and 5-methylcytidine (m 5 C) [138]. This highly sensitive and relatively simple protocol enables small laboratories to compare the abundance of modified nucleic acids across samples.…”

The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation

“…blotting were performed as we previously described 2,25,29 . In brief, following electrophoresis, RNA was transferred onto Hybond N+ positive charged nylon membranes (Cat# RPN303B; GE Healthcare, Little Chalfont, UK) using semi-dry transfer and cross-linked using ultraviolet light.…”