Conrad H. Waddington, a British scientist with an eclectic background in developmental biology, paleontology, genetics, and embryology, first coined the term “epigenetics” in 1942 to describe the then-unknown genetic mechanisms influencing phenotypic outcomes.  Today, those mechanisms have been elucidated in great detail, as a vast array of biochemical modifications that regulate how our genes are expressed is now well-documented. Superficial alterations of the DNA, its transcripts, and the histones it winds around, via the addition or deletion of methyl, acetyl, phosphate, and other chemical groups, can essentially activate or repress gene expression.  Since the 1960s when studies on the epigenetics of chromatin first started, histone and DNA marks, especially DNA methylation, have become the most investigated and most characterized epigenetic modifications to date.  Within a field so heavily concentrated on the intricacies of the epigenome, the equally complex epitranscriptome has undeservedly received far less attention from the epigenetic research community.  That is, until recently.

Dr. Jia Meng, Senior Associate Prof. in Bioinformatics at Xi’an Jiaotong-Liverpool Univ. Credit to Xi’an Jiaotong-Liverpool Univ.

There has been of late an upsurge in appeal surrounding epitranscriptomics and modified RNA. Dr. Jia Meng, Senior Associate Professor in the Department of Biological Sciences at Xi’an Jiaotong-Liverpool University and former Associate Scientist at the Broad Institute of MIT and Harvard, says, “The trend is evident. Studies of RNA modifications and their roles in various diseases appear every week today.  That is totally different from seven years ago when this field just got started and captured people’s attention.  Considering the large number of RNA modification types (greater than 100) and the vast unknowns in this field, it is likely that RNA epigenetics (or epitranscriptomics) will get increasingly popular in the next ten years.”

According to Dr. Kate Meyer, Assistant Professor in the Department of Biochemistry at Duke University School of Medicine, “In just the last six to seven years, there has been a huge increase in the amount of research into the epitranscriptome.  This was sparked initially by studies revealing the prevalence of m⁶A, but quickly expanded to other modifications as well.”

N⁶-Methyladenosine, or m⁶A, is formed when a methyl group is chemically added at the nitrogen-6 position of adenosine residues.   Often referred to as “the fifth RNA base”, m⁶A is the most common and abundant eukaryotic RNA modification, accounting for over 80% of all RNA methylation.  It can be found mainly in mRNA, but is also observed in non-coding species like tRNA, rRNA, and miRNA.

Through interactions with various binding proteins called “readers”, m⁶A affects virtually every facet of ribonucleic acid biology: structure, splicing, localization, translation, stability, and turnover. ¹   Aside from this central role in RNA metabolism, m⁶A plays a part in other physiological processes such as cell differentiation, immunity, inflammation, and the circadian clock. ²   Abnormal m⁶A methylation has been implicated in diabetes, obesity, neurodegeneration, cancer, and other pathologies.  The formation of m⁶A RNA appears to be a co-transcriptional event occurring early on in the RNA lifecycle and is mediated by a multi-protein methyltransferase complex.  The recent discoveries of these methylase “writers” and their associated demethylase “erasers” in mammals uncovered the reversibility of the m⁶A modification, exposing potential therapeutic targets for m⁶A dysregulation-related diseases and generating renewed interest in the epitranscriptome.

Dr. Kate Meyer, Assistant Prof. of Biochemistry at Duke Univ. School of Medicine. Credit to Duke Univ.

“This is a really exciting time for RNA research, because we are seeing that there are a variety of distinct modifications in mRNAs, and we are still in the early days of understanding how these modifications are regulated and what their functions are,” says Dr. Meyer. “Some modifications seem to be low-abundance, whereas others (like m⁶A) are much more prevalent.”

Dr. Meyer’s seminal work in 2012 on m⁶A-modified mRNA led to the development of MeRIP-seq, the first method to detect m⁶A on a transcriptome-wide level. ³   She adds, “I think that as the field continues to develop better and more quantitative methods for measuring RNA modifications, we will improve our understanding of these marks and how they contribute to gene expression.

Investigators examining m⁶A RNA methylation rely primarily on immunoblotting and global profiling techniques, including dot blot, immuno-northern blot, and m⁶A-seq. ⁴   However, these antibody-based methods have their drawbacks, requiring a high starting RNA amount, lacking the single base resolution necessary for precise m⁶A site determination, and displaying cross-reactivity with unmethylated adenosine and m⁶A analogs.

m⁶A-seq requires a large volume of RNA input and sometimes fails to capture a strong enrichment signal in the immunoprecipitation step,” notes Dr. Meng. “In the RNA modification field, it is often necessary to use several orthogonal approaches to validate the results.”

 “m⁶A antibodies have limitations, including cross-reactivity with m⁶A_m and the requirement for high amounts of input RNA for transcriptome-wide profiling studies,” Dr. Meyer points out.  This prompted her lab to come up with a novel antibody-free method for m⁶A detection called DART-seq, an innovative technology that selectively deaminates m⁶A-adjacent cytosine bases, converting them to uracils while leaving unmethylated A-C motifs intact. ⁵  “This allows us to use very low amounts of input material to obtain m⁶A maps.  Most projects in our lab have now transitioned to using DART-seq instead of antibody-based approaches, and we are working to make this method even more sensitive so that we can better detect low-abundance m⁶A sites.”

Despite the limitations of today’s m⁶A analytical techniques, epitranscriptomic research is pushing forward at an unprecedented rate.  For Dr. Meng, considerable improvements upon present methodologies of RNA methylation analysis are still needed: easier protocol, higher reliability (we found that there often exists significant discrepancy among the results obtained from different RNA methylation profiling technologies), lower cost, higher (or even base) resolution, capability of accurate methylation level quantification.”

Dr. Meyer agrees: “I think that a major limitation for m⁶A detection strategies has been obtaining accurate quantitative information.  Methods like SCARLET have been used for measuring the amount of m⁶A on the level of individual sites, but the field is still in need of a reliable method for global m⁶A quantification.  Recently developed strategies such as MazF-dependent m⁶A profiling are very exciting because they not only provide nucleotide-resolution m⁶A maps, but quantitative information as well.  However, MazF can only be used to identify a subset of m⁶A sites.  DART-seq enables simultaneous single nucleotide-resolution m⁶A detection and quantification of the entire transcriptome.”

At the moment, Dr. Meyer’s lab is focused on optimizing DART-seq’s quantitative capabilities in order to establish a “one-stop shop” for both the identification and quantification of m6A sites in any sample.  “Obtaining global measures of m⁶A abundance will be important going forward,” Dr. Meyer envisions,“since dynamic changes to the methylome which involve altered levels of m⁶A are difficult to detect using current antibody-based approaches.”

The majority of RNA methylation research continues to incorporate the use of m6a antibodies for now, with ELISA and MeRIP still being the most popular approaches.  As investigators press ahead to work out the kinks, better-quality antibodies with improved target specificity and minimal off-target binding will be essential for more efficient m⁶A assessment.  There is room for refinement as new technologies and methods emerge for m6A, with a revived sense of excitement not seen since the discovery of 5-hydroxymethylcytosine’s role in epigenetics.

References

1. Zaccara, S. et al. "Reading, writing and erasing mRNA methylation". Nat Rev Mol Cell Biol 20, 608–624 (2019)

2. Hastings, Michael H. “m6A MRNA Methylation: A New Circadian Pacesetter.” Cell, vol. 155, no. 4, 2013, pp. 740–741.

3. Meyer, Kate D., et al. “Comprehensive Analysis of MRNA Methylation Reveals Enrichment in 3' UTRs and near Stop Codons.” Cell, vol. 149, no. 7, 2012, pp. 1635–1646.

4. Zhu, Wei, et al. “Detection of N6-Methyladenosine Modification Residues (Review).” International Journal of Molecular Medicine, 2019

5. Meyer, Kate D. “DART-Seq: an Antibody-Free Method for Global m6A Detection.” Nature Methods, vol. 16, no. 12, 2019, pp. 1275–1280.