By now, you'd think the research community would stop underestimating RNA. After all, RNA—once considered the boring middle child of molecular biology—is full of surprises.
First, there was the discovery of mRNA splicing. Then came long noncoding RNA. And RNAi. And microRNA. And piwi-interacting RNA. So when researchers started to see that not only could RNA assume yet another form but that this form was also incredibly widespread, you'd think journal editors and scientists would be ready … but you would be wrong.
In December 2012, Norman (“Ned”) Sharpless, Deputy Director of the Lineberger Comprehensive Cancer Center at the University of North Carolina School of Medicine, published an article in the journal RNA showing that human fibroblasts contain more than 25,000 circular RNA species arising from 14.4% of the expressed genes.
That study was not the first to demonstrate the existence of RNA circles. As far back as the 1980s, researchers have observed the occasional ribonucleic acid ring, including hepatitis delta virus RNA and transcripts of the sex-determining Sry gene in mammals. However, most of these observations were dismissed as splicing accidents or artifacts of EST libraries. In the past few years, however, a growing body of research, based both on bioinformatic and biochemical data, suggested that circular RNAs were perhaps more abundant than researchers thought—that is, if they remembered RNA circles existed at all.
Still, circular RNAs appeared to be exceedingly rare. But Sharpless’ 2012 data implied that circular RNAs were everywhere, representing a substantial fraction of a cell's transcriptional output. The response from peer reviewers was less than enthusiastic.
“I've been running my lab now for 12 years,” Sharpless says. “I've published in Cell, Nature, NEJM … and I've never had reviews like this. They were nasty. They were really mean.”
Sharpless submited his article to five different journals prior to its publication in RNA, but not before Patrick Brown's team at Stanford University beat him to the punch with a similar study in PLoS ONE. Only three months later, in February 2013, back-to-back manuscripts in the journal Nature officially put circular RNAs on the map (and made headline writers’ day): “Circular RNAs throw genetics for a loop,” wrote Nature; “A circuitous route to noncoding RNA,” was Science's take.
The funny thing is, circular RNAs were hiding in plain sight the whole time. So why were they missed for so long? In a word: Assumptions.That can't be right…
Sharpless circular RNA story began several years ago on the short arm of human chromosome 9. There lies a series of genes implicated in cancer, aging, and atherosclerosis. The locus, Sharpless says, “is one of the strangest … in the human genome.” It encodes 3 tumor suppressor proteins, as well as an exceptionally long 100 kb noncoding RNA transcript called ANRIL that somehow controls expression of the nearby coding genes. Sharpless wanted to know how.
In 2010, his team discovered that ANRIL exists in multiple forms. Some transcripts were in found in the usual linear form, with exons in the expected order: 1, 2, 3, and so on. But a substantial fraction adopted a circular structure—not a splicing lariat, but a fully closed exonic circle that the team called cANRIL. In those molecules, exon 5 followed exon 14, and outward-facing PCR primers positioned at either exon actually yielded a product, which would not happen with a linear molecule.
As Sharpless recalls, at the time even he doubted the truth of those results, attributing the data to sequencing errors. But the postdoc leading the project, Christin Burd (now on the faculty at Ohio State University), persisted, “go[ing] to elaborate lengths to prove to me that they were circles before we could publish.”
Finally, he relented. “I guess I can be just as close-minded as our reviewers were.”
Still, circular or linear, the question remained: What does ANRIL do? Taking a cue from noncoding RNA transcripts of the Xist gene, which bind and recruit epigenetic regulators to the inactive X chromosome, Sharpless guessed ANRIL might serve a similar function at 9p21. But he also wondered if the linear and circular forms might differ in potency—that is, could the circles provide a mechanism to tweak epigenetic regulation? He decided to see if other such circles might also exist in the cell.