A Conversation with Samie Jaffrey.

Dr. Jaffrey: I’m going to talk about our work on RNA modifications and, in particular, methylated adenosine. Most of the mRNA [messenger RNA] in the cell is composed of A, C, G, and U, but a tiny bit—maybe one in every 300–400 adenosines—is methylated, and when it’s methylated on the nitrogen of adenosine, it’s called mA. We normally don’t think of mRNA as containing that many modifications; we think of tRNA [transfer RNA] and other RNAs as having modifications. This was discovered way back in 1974 when scientists were beginning to understand how mRNA capping and other phenomena occur. When they were doing metabolic labeling to study the mG cap on mRNA, they ended up inadvertently discovering that there were actually methyl modifications inside mRNA, and with some clever biochemistry they figured out it was mA. The people who did this were Dawn Kelley and Robert Perry at Fox Chase and Fritz Rottman and other researchers like Jim Darnell. Some of the pioneers of molecular biology were involved in discovering this modification. mAwas known to be low abundance, but even though it was discovered way back then, almost nothing has been known since about what its function could be. It’s very appealing to think that it might be a modification like phosphorylation is for proteins. The potential that mA has some sort of regulatory function has been overlooked, and it’s for many reasons. First, some people didn’t even think that mA was really in mRNA, because when you purify mRNAyou actually have a lot of contaminants and they thought maybe mA could have been from tRNA or ribosomal RNA. But people like Jim Darnell did experiments where he did exceptionally high-purity purification, at least to the ability that they had at the time, and they still were able to see mA. So, there was belief—but still controversy—about whether it was really in mRNA. The other thing is, how do you study mA? mA behaves exactly like adenosine when you do reverse transcription. If you take mRNA and you make cDNA [complementary DNA], which is the reverse transcription step in molecular biology, you’re going to lose any methyl marks that were there. A lot of people abandoned the field and quickly moved into splicing because that was discovered in the end of the ’70s. A lot of this was lost and it wasn’t even in textbooks, but there were a few researchers who stayed on it. Fritz Rottman at CaseWestern was really the pioneer who cloned the enzyme. It’s called METTL3, or methyltransferase-like enzyme 3. Then, as with so many other things in molecular biology the breakthroughs came from yeast and plants. Some scientists knocked out the enzyme in yeast and they found a very remarkable sporulation defect. In plants they found seeds would go to one stage of development and stop. That was Rupert Fray’s paper in 2008. When we saw that paper we were just completely shocked; we never even heard of this modification. I had a postdoc who started shortly thereafter; her name is Kate Meyer and she’s now at Duke. I said, “We need to figure out what is this mA. Is it really in mRNA?Where is it? Which mRNA? All mRNAs?” It could have been like the cap, where every RNA has it, or a tail or it could be selected. We had no idea. But that paper—and it related to seed development—told us that the effects were precise. The cells didn’t just die. The yeast data, which had come out in 2002, really showed us that there was some connection between developmental and cell-fate decisions like sporulation and seed development; they’re all developmental type processes. We wanted to figure out what was going on. That was when we applied for our first NIH grants on this topic. There were mA-binding antibodies that were being developed for other purposes to study DNA methylation in bacteria and things like this. So we got those antibodies and we pulled down mRNA fragments, and it turned out those antibodies bound very specific regions of mRNAs, which are the regions that have mA in it. We were able to identify the transcripts in the transcriptome that have it. And it wasn’t every transcript; it was very specific transcripts. These transcripts tend to have very unusual features. They tend to have huge exons, and we think those exons are the trigger for the methylation. They tend to occur in transcripts that encode regulators of cell fate