The diversity of RNA modification is “a bit of an undiscovered country,” says Christopher Mason, assistant professor of physiology and biophysics at Weill Cornell Medicine, one of the pioneering investigators of RNA epigenetics. Indeed, less than a decade ago, the field didn’t exist. When Mason and Samie Jaffrey’s first paper on mapping the epitranscriptome, identifying methylation of the N6 position of adenosine (m6A) as a pervasive modification found in a significant portion of cellular mRNA, was published in 2012 (together with another work from Israel published at the same time), he says, “We spent probably 20% of the paper trying to characterize the antibody to make sure what we were seeing was real and reproducible.”
But now, more than five years since that study—which was the first to coin the term “epitranscriptome,” multiple companies such as Epigentek, Qiagen, Synaptic Systems, and Abcam offer a variety of antibodies, reagents, and technologies to study the epitranscriptome. “Now, it’s a field where before it was just a collection of ideas,” says Mason, who is also the lead investigator on NASA’S Twin Study of DNA and RNA Methylation Before, During, and After Human Space Travel, which studies how related environmental factors influence chemical changes in RNA and DNA.
“Now, it’s a field where before it was just a collection of ideas…”
And the barely kindergarten-aged field has already begun to demonstrate its significance in human disease. “Research published within the last year has suggested a role for m6A in separating high and low risk leukemias, and we’re seeing its significance in brain cancers,” Mason explains. “RNA modification is also opening up fundamental questions of virology: to date, every single RNA we have examined has some kind of RNA modification, some m6A or base dynamic we’ve never seen before.”
Last year, Mason and collaborators at Duke mapped the presence of N6-methyladenosine tags in viruses including hepatitis C, Zika, dengue, West Nile, and yellow fever, which affect their ability to infect cells.
“We know of about 120 epigenetic markers on RNA so far, but there could be 300 or a thousand. The total plasticity of RNA biology is still being discovered,” Mason adds. “The next big question is whether it will be a lever for some therapeutic actions.”
Among the key challenges in moving the field forward, says William Lee, vice president of operations at Epigentek, is establishing robust and useful methods, such as RNA modification sequencing, to identify RNA modifications including m6A at the single base-resolution level.
“MeRIP-seq in general gives a resolution at ~100 bp scale for m6A identification but cannot reach single base-resolution level,” he observes. “Recently an improvement for sequencing resolution is to use anti-m6A antibodies to induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA crosslinking and reverse transcription, which can make mutational signatures and help to map m6A residues at nucleotide resolution.” Related research was published by Jaffrey, Greenberg-Starr Professor in Weill Cornell’s Department of Pharmacology, and Mason in 2015.
Chuan He, John T. Wilson Distinguished Service Professor in Chemistry and director of the NIH Center of Excellence in Genomic Science at the University of Chicago, was a key early investigator of m6A. In 2010, he proposed that RNA methylation is reversible much like DNA methylation, and in 2011, he discovered and published the first RNA demethylase that largely sparked the recent wave of epitranscriptome work. “We need a quantitative method for RNA methylation sequencing equivalent to bisulfite sequencing for DNA methylation,” He explains. “You want to know exactly where the modification occurs, and is it 25% modified at this site or 50%? That’s our first challenge.”
Methods in flux
Current profiling methods are also limited by the requirement of a large amount of material for performing experiments. “That essentially eliminates the possibility to study, for example, specific neurons,” He says. “m6a methylation plays a critical role in early development of nervous system functions, where you have to sort out different types of neurons, that level of information can be challenging to obtain with current methods. It’s difficult to perform quantitative sequencing with limited material, including biopsy samples.”
Biocompare’s Gene Expression Search Tool
Find, compare and review gene
expression tools from different suppliers Search
RNA’s complexity makes it inherently more difficult to work with than DNA, agrees Samuel Rulli, global product manager for Qiagen. “You need a way to isolate the RNA—a way that changes with modifications—and then a way to work with it downstream. Starting with a high-quality sample helps you find something that’s reproducible rather than an artefact. That’s critical, especially when you’re in the early stages of a field like this: you need consistency across assays to be able to see these needle-in-a-haystack things.”
Another major challenge, He says, is to read out the presence of different modifications on one RNA. “With current methods, whenever you work with one cell, you get an average. The question is, do the modifications work synergistically, or do they not care about each other? It would be nice to have a method where you could examine every single mRNA, read out all the modifications occurring—not just m6A—and see how they coexist. If we can solve these problems, it opens up enormous research possibilities for this field.”
Nanopore direct RNA sequencing may begin to address some of these issues, says Mason. “A lot of what we’ve done historically is that we’ve inferred modified RNA states, what we think is there, by an antibody or binding or chemical assay. We haven’t done direct RNA sequencing until just this past year. With Nanopore, we sequence the RNA directly, rather than using reverse transcription, and measure chemical composition and electrical change. For the first time ever, we can directly measure single molecule RNA modifications: the beginning and end of the entire molecule, what modifications are present, what isoforms are present.” His group now has several papers in press using this technology.
“Just within the past year, I think, people have really started to realize that the epitranscriptome fundamentally affects gene expression,” He notes. “The questions of how it does that in different cell types and developmental stages, how the specificity is achieved and how is it regulated, are just beginning to be explored. To me, the field has just started.”