Quantifying N6-methyladenosine broadly and specifically with the new eTAM-seq method

N6-methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification in higher eukaryotic species. It has broad and heterogeneous functions depending on the mRNA, the specific site within an mRNA, and across cell types. m6A offers a layer of regulation to mRNA processing, translation, and decay without changing the genetic code—it could be called a component of the mRNA epigenome. 

eTAM-seq provides a new scale of flexibility for m6A identification 

Thus far, methods to detect m6A rely mostly on antibodies to broadly immunoprecipitate methylated RNA followed by deep sequencing. The current strategies are limited by required significant inputs, antibody specificities, crosslinking efficiencies and artifacts, biases in sequence context, secondary structure, and generally complicated workflows. However, a recent paper in Nature Biotechnology from a group at the University of Chicago describes a new method for detecting and quantifying m6A. Their method, called eTAM-seq, has the advantage of detecting the modification both broadly with deep sequencing and specifically when paired with a simple workflow and Sanger sequencing.   

The authors were inspired by the concept of bisulfite sequencing, which is a method used to detect DNA methylation. However, the authors wanted an enzymatic-based technique specific for m6A detection and quantification. Building off previous work that examined the efficiency of adenine deaminases, they identified a specific adenine deaminase enzyme with the qualities sought including minimal context dependency, efficient (complete) and specific deamination, maintenance of RNA integrity, and effectiveness in mild reaction conditions. They paired this enzyme, termed TadA8.20, with RNA-sequencing to create eTAM-seq. In eTAM-seq, the sample is first treated with TadA8.20 for global deamination resulting in the unmethylated adenosines being converted to inosines. Inosines pair with cytosine; after reverse transcription, they are read as guanosines. Therefore, a persistent A signal represents an m6A-modified residue.  

eTAM-seq technique validated with synthesized oligos  

The researchers performed several control experiments throughout the paper to validate their new method. In their first set of experiments, they tested the efficiency of TadA8.20 to deaminate in vitro transcribed synthesized oligos. The in vitro transcription reactions used N6-methyl-ATP from TriLink Biotechnologies. The results of these experiments showed nearly 100% specific conversion for unmethylated adenosines and almost no conversion to guanosine from m6A residues. These oligo experiments demonstrated that the TadA8.20 enzyme was useful for deamination and that eTAM-seq could correctly identify and quantify m6A-modified bases. 

Global m6A patterns examined in HeLa cells 

Following this initial proof of concept, the authors performed eTAM-seq paired with RNA sequencing in HeLa cells. Using HeLa cells allowed the researchers to compare eTAM-seq results to datasets with the most commonly established technique, MeRIP-seq. Through this profiling experiment, the paper confirmed the presence of a context motif previously noted for m6A, had an 89% overlap with MeRIP-seq datasets and verified the level of m6A in 20 different specific mRNAs. 

After establishing eTAM-seq as a global m6A profiling technique in HeLas, the group tested eTAM-seq in a more challenging biological context: mouse embryonic stem cells (mESCs). While they found a slightly lower overlap between eTAM-seq results in mESCs vs. Me-RIP data (52-74% overlap) than in HeLas, there was still considerable consistency between techniques. They went on to examine specific pluripotency factors in their mESCs, validating the methylation status of these mRNAs with known literature and MeRIP-seq data. The experiments in mESCs show that the technique is successful with a challenging cell population and context. 

Site-specific detection with eTAM-Sanger 

Finally, the paper described a second way to use eTAM-seq: for site-specific identification and quantification of m6A when paired with Sanger sequencing. For this protocol, the RNA is polyA-deadenylated, fragmented, has adapters ligated to the fragments, is TadA8.20 treated, reverse transcribed, amplified, and sequenced. Data showed minimal deviation from whole transcriptome results (median deviation of 5.9%) and proper stoichiometry quantification to within 10% in 15 of the 18 sites examined. Importantly, using eTAM-Sanger, the authors showed that eTAM-seq was equally specific for m6A detection with extremely low levels of input RNA: traces successfully identified the modification from 250 pg of total RNA, which is equivalent to the amount in 10 cells.  

As Sanger sequencing is less accurate with sequences containing high guanosine contents, the paper also demonstrated that eTAM-seq is compatible with amplicon deep sequencing. Therefore, eTAM-seq is a specific and quantitative method for determining sites of m6A when paired with whole-transcriptome sequencing, amplicon deep sequencing, or Sanger sequencing. 

Conclusion 

In conclusion, eTAM-seq allows researchers to investigate the m6A status in their experimental setup on either a global transcriptome or a single-site scale. Additionally, eTAM-seq is possible to perform with a simple molecular biology workflow when done with Sanger sequencing. As such, eTAM-seq provides a powerful new tool to understand the functional consequences of m6A modifications in various cellular contexts.  

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Article reference: Xiao YL, Liu S, Ge R, et al. Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination. Nat Biotechnol. 2023;10.1038/s41587-022-01587-6