Molecular Medicine Israel

Uncovering Functions of Circular RNAs

Recent research has revealed many surprises about circular RNAs, from findings that they are translated in vivo to links between their expression and disease

NA comes in many shapes and sizes. Over the past few decades, researchers have characterized at least two dozen different RNA varieties beyond the textbook classics. But a type of RNA that long flew under the radar due to its designation as a molecular mishap is now taking center stage.

Circular RNAs (circRNAs), or simply “circles” to many researchers, are just what they sound like: nucleotides of RNA arranged in a closed loop. Much about the function of these molecules remains a mystery, but for some time, at least one thing seemed clear: unlike linear messenger RNA (mRNA), circles were not translated into proteins in living organisms. “When you have any type of RNA, you wonder whether it’s translated,” says Sebastian Kadener, a molecular biologist who has spent the last few years researching circRNA at the Hebrew University of Jerusalem. Despite reporting the presence of one or more protein-coding exons in many circRNAs, multiple studies in the past few years failed to find evidence of the molecules associating with ribosomes in vivo. If circles were doing anything at all, many researchers agreed, they must be doing it as untranslated RNA.

A little over a year ago, however, Kadener and his colleagues detected something that would upend that assumption: an average-size (37 kilodaltons) protein encoded by a naturally occurring circRNA in Drosophila. Along with collaborators in Germany, Kadener’s group used a method known as ribosomal footprinting to detect RNAs being actively translated in extracts from fly heads. Not only did the researchers discover more than 100 different circRNAs—ranging from around 300 to more than 2,000 nucleotides in length—apparently associating with ribosomes in the cells, they also identified a protein that, based on its sequence, could only have been translated from one of these circles, not from a standard linear transcript. “We could see the protein by Western blot,” Kadener says. “It was being expressed in the synapses of flies.”

Kadener’s work was published earlier this year,1 back-to-back in Molecular Cell with another group’s study—on human and mouse cells—that had simultaneously come to the same conclusion: translation of circRNAs can and does occur in living cells.2 For now, neither group has any hint of the function of these proteins, or of how common circRNA translation really is, but “you can imagine that it has some biological importance,” Kadener notes. RNA researcher William Jeck, currently a fellow at Harvard Medical School, agrees. Many scientists had “written off translation,” he says. “This is extremely exciting evidence that other circles may produce peptides that may be biologically relevant. . . . It’s really changed the paradigm.”
NA comes in many shapes and sizes. Over the past few decades, researchers have characterized at least two dozen different RNA varieties beyond the textbook classics. But a type of RNA that long flew under the radar due to its designation as a molecular mishap is now taking center stage.

Circular RNAs (circRNAs), or simply “circles” to many researchers, are just what they sound like: nucleotides of RNA arranged in a closed loop. Much about the function of these molecules remains a mystery, but for some time, at least one thing seemed clear: unlike linear messenger RNA (mRNA), circles were not translated into proteins in living organisms. “When you have any type of RNA, you wonder whether it’s translated,” says Sebastian Kadener, a molecular biologist who has spent the last few years researching circRNA at the Hebrew University of Jerusalem. Despite reporting the presence of one or more protein-coding exons in many circRNAs, multiple studies in the past few years failed to find evidence of the molecules associating with ribosomes in vivo. If circles were doing anything at all, many researchers agreed, they must be doing it as untranslated RNA.

A little over a year ago, however, Kadener and his colleagues detected something that would upend that assumption: an average-size (37 kilodaltons) protein encoded by a naturally occurring circRNA in Drosophila. Along with collaborators in Germany, Kadener’s group used a method known as ribosomal footprinting to detect RNAs being actively translated in extracts from fly heads. Not only did the researchers discover more than 100 different circRNAs—ranging from around 300 to more than 2,000 nucleotides in length—apparently associating with ribosomes in the cells, they also identified a protein that, based on its sequence, could only have been translated from one of these circles, not from a standard linear transcript. “We could see the protein by Western blot,” Kadener says. “It was being expressed in the synapses of flies.”

Kadener’s work was published earlier this year,1 back-to-back in Molecular Cell with another group’s study—on human and mouse cells—that had simultaneously come to the same conclusion: translation of circRNAs can and does occur in living cells.2 For now, neither group has any hint of the function of these proteins, or of how common circRNA translation really is, but “you can imagine that it has some biological importance,” Kadener notes. RNA researcher William Jeck, currently a fellow at Harvard Medical School, agrees. Many scientists had “written off translation,” he says. “This is extremely exciting evidence that other circles may produce peptides that may be biologically relevant. . . . It’s really changed the paradigm.”
See “Circular RNA Surprise”

Because microRNAs are involved in regulating translation—by binding to specific mRNAs, they trigger degradation of transcripts through a process known as RNA interference (RNAi)—Hansen and his colleagues speculated that the findings might indicate a general role for circles in regulating gene expression. “At that time, we were searching for other circular RNAs, but pipelines for detection weren’t really established,” Hansen tells The Scientist. When they found similar roles for two circRNAs, “we didn’t know, but of course we hoped that it could be a general thing, that circular RNAs would emerge as [regulators] of these micro-RNAs—it made a lot of sense.”

But researchers now believe that most circles are unlikely to act as microRNA sponges. As the number of known circRNAs has climbed into the thousands, only a handful of sponges have been identified. And a 2014 study using computational methods to predict sequences likely to make good sponges identified only a few other candidates.9 The authors of that study “made a strong case that it wasn’t a general function,” says Salzman. Instead of sponging, circRNAs may be engaging in other types of microRNA interactions, Hansen notes. “I think [circles] could have more profound effects in terms of stabilizing [microRNA], or directing it to certain parts of the cell—although that’s of course hypothetical at the moment.”

CircRNAs also appear to associate with proteins, suggesting another suite of potential regulatory functions. For example, researchers recently showed that a circRNA produced by the Foxo3 gene (called circ-Foxo3) interacts with proteins involved in cell proliferation, including a key cyclin-dependent kinase and one of its inhibitors, suggesting a role in the cell cycle. And while most exon-containing circles accumulate in the cytoplasm, those that retain introns are often found in the nucleus, where they encounter proteins involved in transcription. In 2015, scientists in China showed that a group of exon-intron circRNAs promoted transcription of their parent genes via interaction with RNA polymerase II.10 Other studies have shown circles interacting with different RNA-binding proteins as well, including proteins now linked to circRNA biogenesis, such as Muscleblind and Quaking, and Argonaute proteins, well-known for their participation in RNAi-based gene regulation.

There’s the possibility that the regulation of circRNA biogenesis itself constitutes a function, too. Because each RNA transcript can be either linear or circular, but not both, upregulating circularization could act as a mechanism to reduce the proportion of linear mRNA generated from a particular gene. A recent study by Rajewsky and Kadener showed that strong competition between circularization and linear splicing can occur, most likely due to overlapping dependence on the same splicing machinery—although the extent to which it constitutes a function per se is still unclear.11

With the recent description of in vivo translation of circRNAs comes an entirely new dimension of possible functions—one that researchers are only beginning to explore. “I’m sure that people are now going to be looking to see when these proteins are produced, where these proteins are produced, et cetera,” says Kadener, adding that his team plans to further investigate the role of translated circRNAs in Drosophila brain function. “You can imagine so many hypotheses of what this translation might mean. . . . The protein made by the circle could modulate other proteins, for example. It opens a lot of possibilities.”

Like all of the speculation about circRNA function, though, hypotheses about translation will have to be pursued with a healthy dose of skepticism, notes Wilusz. “It’s certainly a very attractive idea,” he says. “It would make sense in some way, that if you’re making [a circRNA] from a protein-coding gene, you should make a protein. But there’s a lot more work that needs to be done to prove that the proteins are being produced at high levels—or even do anything.”

Putting circles to use
As RNA researchers continue to explore circles’ possible functions, multiple labs have discovered that circRNA expression levels vary substantially with disease, leading to growing interest in how these molecules might be harnessed for diagnosis and treatment. Certain circRNAs are up- or downregulated in cancers of the skin, liver, bladder, larynx, and stomach, to name a few. And it’s not just cancer; abnormal expression of several circRNAs has also been linked to cardiovascular disease and to neurological disorders such as Alzheimer’s and Parkinson’s.

CDR1as, for example—one of the original microRNA sponges and the best-studied circle to date—is linked to a number of diseases, in several cases via its sequestration of miR-7. A well-characterized tumor suppressor, miR-7 inhibits cell growth, and its loss is associated with poor prognosis. “High expression of CDR1as is not very good in terms of cancer,” Hansen explains, “because it inhibits the microRNA that would normally protect from cell proliferation.” Looking beyond cancer, researchers in China reported in 2015 that overexpression of miR-7 in pancreatic islet cells led to impaired insulin production and diabetes in mice—an outcome the team suggested was normally kept in check by the sponging activity of CDR1as.12 And reduced expression of CDR1as in the hippocampus has been associated with Alzheimer’s disease.

Another disease-linked circular RNA, circTCF25, also appears to act as a microRNA sponge. Expressed at high levels, circTCF25 downregulates two microRNAs, leading to cancer cell proliferation in vitro and in vivo in humans—mechanisms that could explain the link between high circTCF25 levels and bladder cancer. And earlier this year, researchers described a complex pathway in which peptide-binding circ-Foxo3—downregulated in several cancers—regulates proteins involved in cancer cell death. The team showed that through interactions with several peptides, circ-Foxo3 increases levels of its parent gene’s protein, Foxo3, which can trigger apoptosis in tumor cells.

These glimpses into circRNA’s role in disease have sparked interest in exploiting the molecules as potential therapeutic targets. A study published earlier this year noted that silencing CDR1as using specially-designed short hairpin RNAs (shRNAs) inhibited proliferation and invasiveness of colorectal cancer cells in culture. And a team at Mount Sinai School of Medicine in New York used similar methods to target ciRS-E2, a circle consisting of a single exon that is highly expressed in cancers such as leukemia and melanoma. The group reported that shRNA treatment dampened ciRS-E2 expression by more than 80 percent in cultured cancer cells, and resulted in significantly reduced proliferation.

For now, though, while functions for the vast majority of circRNAs remain unclear, many labs are focused on exploring the more immediate goal of using circles to classify and monitor diseases with which they are associated. For example, “we’re all very interested in trying to find ways to divvy up tumors into different categories of risk and potential response to therapy,” says Jeck. “CircRNAs do have one really nice feature, and that’s that they are stable. That means if you can get a good sample of cells, you have a really good shot at identifying [them].”

Indeed, researchers recently found that circRNAs are present in circulating extracellular vesicles such as exosomes, and could in some cases provide more information about gene expression in healthy and unhealthy cells than their linear counterparts in easily accessible human fluids. In a 2015 study of blood-borne circRNAs, Rajewsky’s lab discovered that detecting the circular transcripts served as a more faithful proxy for the expression of hundreds of genes than classical mRNA-specific assays.13 “We would not ‘see’ these genes, so to speak, by normal RNA expression,” he says. “So circRNAs could be molecules that tell you something about development or disease that normal molecules do not.”

Specific, circRNA-based biomarkers for several diseases have already emerged from retrospective analyses of patients. In January, researchers described a combination of two circular RNAs, hsa_circ_0124644 and hsa_circ_0098964, that detected coronary artery disease with a specificity and sensitivity rivaling current methods, while presenting a cheaper and more convenient alternative. And other studies in the last two years have highlighted specific circular biomarkers for several cancers, including liver, stomach, and colorectal. Now, these candidates must be validated in studies that predict disease outcome, says Jeck. “There have been a lot of retrospective analyses, and that’s all well and good,” he says. “But I think the next step is to see if people can use circRNA expression in a prospective manner. That would be very exciting and potentially very useful.”

Of course, how circRNAs come to be understood in the lab and possibly one day used in the clinic remains to be seen, as the study of these looped molecules represents an area that’s still young. But if the past five years are any indication, the study of circRNAs is rapidly ramping up. “What’s amazing to me is how fast this field has grown,” says Wilusz, whose lab supplies plasmids expressing circRNAs to other research groups and has recorded a dramatic uptick in requests in the last couple of years. “It’s really taking off.”

Rajewsky, whose group is now focusing on circRNAs’ interactions in the brain, agrees that the best is very much ahead. “We’re really just at the beginning of an exciting journey,” he says. “It doesn’t happen often in molecular biology that you find such a fundamentally new phenomenon.”

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