“It took a lot of head-scratching, but eventually [we] realized that the data was consistent with a model where the exons were circularized.”
Labmates largely ignored her findings, she says, suspecting she had made a programming error. But when she showed the results to Brown, he was hooked. “Many people might have discouraged us from pursuing it, but he always thought that it was a very interesting and worthwhile phenomenon to get to the bottom of— which I just think reflects [Brown's] amazing scientific intuition.”
John Rinn, a noncoding RNA expert at Harvard Medical School, seconds that point. “That guy's a Jedi. He is Yoda. …Pat Brown has constantly had a profound impact in understanding gene regulation.”
Salzman and colleagues Chuck Gawad and Peter Wang eventually demonstrated that hundreds of genes produce circularized isoforms, some of which they validated experimentally using RNase R digestion and outward-directed PCR. The team submitted their findings to PLoS ONE in November 2011, and the article was published the following February.
For Salzman, circular RNAs are not merely molecular curiosities. Their circular structure, she says, “has implications for essentially everything that we know or believe about RNA transcripts,” from how they are spliced and exported from the nucleus, to how and if transcripts are translated. “It makes a big difference.”Everything old is new again
Yet it was back-to-back papers in Nature that really elevated circular RNAs into the scientific community's consciousness in early 2013. Why? These studies were the first to ascribe an actual function to one of these molecules.
The first report, from Nikolaus Rajewsky's lab at the Max Delbrück Center for Molecular Medicine in Berlin, used bioinformatics to identify 1950 circular RNAs in human leukocytes, 1903 in mouse brains (81 of which were also found in the human data set), and 724 from C. elegans. Rajewsky's group then biochemically characterized a subset of these molecules, including a circular form antisense to the human CDR1 gene, which they called CDR1as. A second team, led by Jørgen Kjems at Aarhus University, Denmark, focused specifically on this molecule.
This was not Kjems’ first time working on CDR1as; he actually first described the molecule (which he calls ciRS-7) in 2011. At the time, his team was exploring the hypothesis that microRNAs could repress transcription by acting at the DNA level. In their search for candidate genes, they discovered that the CDR1 promoter contained a perfect match to miR-671. “But the funny thing was, it was a match to the antisense direction,” Kjems recalls—that is, to the strand opposite the CDR1 coding sequence. “But that of course doesn't matter if you look at DNA effects.”
The team spent months chasing down leads, looking at DNA methylation and histone modification patterns. Everything came up negative. So, they shifted tack; maybe miR-671 worked in the usual way, but on an antisense transcript. That transcript was incredibly easy to find. “This transcript could be the highest expressed gene in all the neurons in the brain,” Kjems explains, “it's extremely highly expressed.”
But as they studied it, Kjems also realized the transcript was odd. It “seemed to behave really kind of weirdly on a gel”, running slower than its size suggested. For many, maybe most transcription biologists, the significance of that finding might have been lost. But Kjems was in a unique position to recognize its import.
Thirty years earlier, as a graduate student, he had discovered one of the first circular RNAs, an intron from a thermophilic archaebacterium. He wrote a couple of papers on the topic, left for a postdoc with Phil Sharp at MIT, and never looked at circular transcripts again. But when the ciRS-7 data started rolling in, he says it was like déjà vu. “My senses were open for the possibility that it could be a circular RNA.”
In 2011, the team published that fact in EMBO, along with the observation that ciRS-7 could be cleaved and degraded by miR-671, the first documented case of such a cleavage event in a mammalian transcript.
ciRS-7's story doesn't end here though. ciRS-7 enhances CDR1 expression, probably by aiding in nuclear export, according to Kjems. Unusual among eukaryotic genes, CDR1 contains no introns, which can complicate nuclear export. But it has a second function, which both Kjems and Rajewsky's teams explored. The 1500 bp molecule contains some 70 partial binding sites for a second microRNA, miR-7. Initially, Kjems says, they noticed the repetitive sequence in the antisense message, but attributed that to a repeating amino acid sequence in CDR1 itself. Now he believes he had the story backwards; that is, the protein sequence repeats because the antisense message has multiple microRNA binding sites. “This is how we can actually be a bit fooled,” he says.