Overview of Sequencing Methods for RNA m6A Profiling

Introduction to N6-methyladenosine (m6A)

The post-translational modifications to RNA that can affect a variety of biological processes have gained acceptance as a dynamic epigenetic mark. The most common and abundant alteration in messenger RNA, N6-methyladenosine (m6A), has been related to a host of effects on mRNA fate. Its distribution and functions have been revealed after a thorough investigation.

N6-methyladenosine (m6A) is a common RNA methylation modification that accounts for 0.1 to 0.4 percent of all adenosine. The methyl-transferase complex, demethylase, and corresponding reader all work together to control the dynamic and reversible modification process. M6A affects multiple biological processes, including neuronal development, cell transition, immune response, DNA damage response, and tumorigenesis, as a result of post-transcriptional methylation. As a result, accurate detection of m6A is critical for understanding its complex relationship with human development and common diseases, and it has emerged as a key research focus in the RNA field and epigenetic modification.

Figure 1. Schematic diagram of the m6A-seq protocol. (Dominissini, 2013)

miCLIP-m6A Sequencing

MiCLIP-m6A-seq (m6A individual-nucleotide resolution crosslinking and immunoprecipitation sequencing) is a next-generation sequencing (NGS)-based method for detecting m6A, a common and abundant RNA methylation modification found in a variety of transcripts. In this method, ultraviolet light is used to cross-link mRNA with the m6A antibody, and the antibody cross-linked RNA fragment is purified and transformed into a cDNA library. The circularized cDNA strand is then re-linearized, amplified, and sequenced. The precise positions of m6A throughout the transcriptome are then determined by analyzing crosslink-induced mutations and truncations introduced during reverse transcription. MiCLIP-m6A-seq uses single-nucleotide resolution to map m6A locations in the transcriptome, effectively revealing m6A-containing RNA and RNA-binding proteins.

MeRIP Sequencing

MeRIPseq employs an antibody that detects and characterizes N6-methyladenosine (m6A) in RNA with pinpoint accuracy. Because the 3'UTR of mRNA is involved in mRNA stability, localization, and translation, as well as the binding of RNA-regulatory proteins to these regions, the presence of m6A in this region suggests that m6A may have an impact on RNA metabolism and gene expression. Inhibition of the enzymes involved in RNA methylation has also led to new insights into the biological implications of this modification. As a result, identifying and characterizing modified bases in RNA may help researchers better understand biological pathways.

Photo-crosslinking-assisted m6A sequencing (PA-m6A-seq)

PA-m6A-seq (photo-crosslinking-assisted m6A sequencing) is a next-generation sequencing (NGS)-based method for detecting m6A in its entirety. RNA methylation can be found in a variety of transcripts. It can precisely map m6A in a sample transcriptome. It can precisely map m6A in a sample transcriptome. By injecting 4-thiouracil (4SU) into the growth medium, the technique converts 4SU to RNA. Under 365-nm ultraviolet light, recovered m6A-containing RNA is cross-linked to the anti-m6A antibody after m6A immunoprecipitation. RNase T1 then digests the cross-linked RNA to about 30nt, allowing for efficient sequencing. Under 365-nm ultraviolet light, retrieved m6A-containing RNA is cross-linked to the anti-m6A antibody after m6A immunoprecipitation. RNase T1 then digests the cross-linked RNA to about 30nt, allowing for efficient sequencing.

References:

1. Körtel N, Rücklé C, Zhou Y, et al. Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning. bioRxiv. 2021 Jan 1:2020-12.
2. Zhang H, Shi X, Huang T, et al. Dynamic landscape and evolution of m6A methylation in human. Nucleic acids research. 2020 Jun 19;48(11).
3. Liu H, Begik O, Lucas MC, et al. Accurate detection of m6A RNA modifications in native RNA sequences. Nature communications. 2019 Sep 9;10(1)
4. Dominissini D, Moshitch-Moshkovitz S, Salmon-Divon M, et al. Transcriptome-wide mapping of N 6-methyladenosine by m 6 A-seq based on immunocapturing and massively parallel sequencing. Nature protocols. 2013 Jan;8(1).