Conceptually the secret of life is pretty straightforward. It is best revealed by an analogy. Think of cells as factories. Each carries a manual with instructions for constructing machines, 25,000 or more in the case of humans. Each machine’s assembly is directed by the text of a chapter, one chapter for each machine to be made. To actually synthesize a machine, the instructions in a chapter are copied and sent off to giant fabricating devices, that carry out their assembly.
That’s all there is to it. One just has to substitute biological entities into the analogy where appropriate, DNA is the instruction manual, genes are chapters in it, and RNA molecules are the copies of individual chapters. Proteins are the machines whose structure is dictated by the RNA instructions, and ribosomes are the giant devices that build them.
That’s the big picture. It’s easy to lose sight of, particularly as one immerses themselves in the morass of details, complications, and exceptions that characterize molecular biology.
Let me begin by disclosing the basis for an additional complication: multicellular organisms have over 200 cell types, each with a different population of proteins (machines in the analogy). This observation strongly suggests that there has to be a mechanism for specifying which chapters are copied in the various cell types to specify the protein population in each. There is. There’s a group of machines (proteins) whose job it is to direct the copying apparatus to different chapters. This process is called gene regulation.
It should be clear that this extra bit of detail makes the secret of life vastly more complicated. These “regulatory machines”, like all the others, must themselves be made by copying the appropriate chapters from the book. But how does the cell decide which regulatory machines to make? The answer: other regulatory machines. And so on, ad infinitum, or so it seems. That’s hairy.
But that’s not the least of it. It turns out that determining which chapters to read is only a part of the complex operations controlled by the regulatory apparatus. Here are some others.
I came across an additional means of gene regulation, one of which I was unaware, the other day while browsing “Science”, the journal of the American Association for the Advancement of Science. The regulation involves a change to the RNA copy of the gene, a minor alteration of one of RNA’s letters.
Making modifications to the RNA copy of a gene has been a process that has been recognized for almost fifty years. It’s well known that an RNA copy, a transcript, routinely has its starting end “capped”, its trailing end filled with multiple copies of the letter “A”, and parts of its interior cut and thrown away (you can find out more about these processes under the heading “RNA processing” using any search engine). What is less well known, are the subtle changes to the various “A” bases in RNA copies that I describe below and which I learned about in Science magazine.
Take a look at the figure. It shows the alteration in the “A” base that I’ve been alluding to. The change is shown in red for emphasis because it may not be obvious at first glance. It’s a subtle alteration, the substitution of a methyl group for a hydrogen, analogous to changing the font weight (to bold or italic, for example) in a section of text. It does not change the meaning of the words, but can make a difference in emphasis. I’ll refer to the modified base as m6A, its nickname.
The methylation of some “A”’s in RNA was discovered decades ago. What wasn’t known are the consequences of the alteration or the players involved in adding or removing the methyl group. There’s been a lot of recent activity in those areas recently.
Writers and Erasers
The increased activity was initiated by a discovery by a group of scientists from the University of Chicago (Jia et al., Nature Chemical Biology 7:885–887 (2011)) that an enzyme with the bizarre name of “fat mass and obesity-associated protein (FTO)” acted as an “eraser” to remove the methyl group (CH3) from m6A. The FTO protein, as its name implies, seems to play an important role in metabolizing fats. It, and another eraser, ALKBH5, seem to be responsible for removal of m6A from mRNA’s. In addition to erasers, m6A “writers” have been discovered that add m6A to RNA. It’s been estimated that these writers are responsible for the 3 to 5 m6A’s found per molecule of mRNA.
Subsequent studies showed that m6A was found predominantly in certain parts of messenger RNA, particularly near its termini. It’s also found more often in areas that will be spliced out of pre-mRNA transcripts. Both the localization of the modifications, and the fact that there are specific enzymes dedicated to their addition and removal suggested that m6A might somehow lay an important role in regulating aspects of mRNA behavior.
Today we know that besides erasers and writers, there are “readers”, that detect the presence of the modified RNA. They do so in three ways. Some proteins are known that bind preferentially to modified RNA’s, specifically recognizing m6A on the molecules. Some bind only to RNA’s in which the m6A modification has been removed. And some proteins detect m6A modification not by binding to the methylated base, but by virtue of the fact that m6A changes the overall three dimensional structure of of the modified RNA’s.
The key question: What processes do these readers affect? In other words, what is the effect of this specific methylation of RNA? The answer in a word: it’s complicated. In fact, a recent review article (“It's complicated... m6A-dependent regulation of gene expression in cancer”, Christina M. Fitzsimmons, Pedro J. Batista,. BBA - Gene Regulatory Mechanisms 1862: 382–393 (2019)) acknowledges that in its title. The authors emphasize that m6A modification affects many processes, including RNA stability, RNA processing, RNA translation efficiency, RNA storage, and RNA transport from the nucleus to the cytoplasm. But the main focus of their paper is the role that m6A plays in cancer.
Patients with acute myeloid leukemia have been shown to have increased levels of FTO. In addition, higher amounts of ALKBH5, the other major demethylase, are correlated with poorer prognosis for patients with glioblastoma, a brain cancer. In breast cancer, the low oxygen environment found in most tumors causes the over expression of ALKBH5, which, in turn, demethylates, and thereby stabilizes a specific mRNA that promotes cancer cell growth. Overall, Fitzsimmons and Batista’s paper lists 12 different cancer types that are influenced by mutations in erasers, writers, or readers. It’s been known for some time that m6A is essential for maintenance of the differentiated state, and when it is lacking cancer is more likely to occur.
One observation that further points to the importance of m6A is that it, and the enzymes that control and recognize it, are found in all eukaryotes assayed to date. Moreover, mutations in m6A writers are lethal in many organisms, indicating that this RNA modification is essential for life. Among the processes affected by reader, writer, and eraser mutations are circadian rhythms, neural development, learning, and progression through the cell cycle.
These are early days for the study of m6A. But it’s already clear that this previously neglected area of gene regulation is no longer being neglected. Expect much more attention ahead.