Soaking up the Competition – Circular RNAs as Molecular Sponges for miRNAs
Numerous families of non-coding RNAs have been identified in mammalian cells, including lncRNA, miRNA, siRNA, and piRNA. Another type of RNA called circular RNA (circRNA) has remained a mysterious class, as the exact function of these molecules has not been described. Usually, circRNAs result from spicing events and have been identified in plants, animals, and archaea. Recently, Memczak et al. published a paper in Nature reporting evidence that circRNAs tend to show specific expression profiles that are associated with different tissues and developmental phases and function as post-transcriptional regulators.
The authors began their investigation of circRNAs by creating a new algorithm to locate circRNAs in any region of genomic DNA. They estimated that their system has a sensitivity of greater than 75% and a false discovery rate of less than 0.2%. Using this algorithm, thousands of circRNAs were predicted to exist in mouse and human cells. The predictive method was validated by the experimental screening of randomly chosen circular RNAs from HEK293 cells. The circRNAs were reverse transcribed and evaluated by quantitative PCR with divergent primers and finally Sanger sequencing. 83% of the predicted circRNAs from HEK293 cells tested were in fact circular, indicating that circRNAs are widely expressed in human cells, in agreement with previous reports.
The authors also found that the circRNAs were on average at least 10 times more resistant to RNAse R activity when compared to the linear control GAPDH in vivo (RNAse R is an exonuclease that digests linear RNA), and circRNAs persisted at high concentrations despite a transcriptional block lasting 24 hours. These results indicate that the circular nature of these RNAs offers a unique degree of protection that makes them extremely stable compared to linear RNA.
The functional investigation of circRNAs began with the hypothesis that the circRNA could act as a sponge to bind and sequester miRNA in a sequence-specific manner. By data mining the list of circRNAs predicted by the algorithm, the authors were able to screen each circRNA sequence for miRNA matches and found that repetitive sequences matching miRNA were localized at a significantly higher density in circRNAs relative to coding sequences or 3’ UTR sequences. For example, an RNA that is antisense to the cerebellar degeneration-related protein transcript-1 (CDR1as) is a known human circRNA and was found to contain 74 miR-7 seed matches.
Furthermore, the authors were interested in the potential effect of circRNAs on proteins that interact with miRNAs. They chose to investigate the human Argonaute (AGO) protein, which directs miRNAs to coding mRNAs to repress translation and found that AGO interacts with CDR1as with high density. These results further support the role of circRNAs as inhibitors of miRNA mediated suppression of gene expression. The circRNA CDR1as was found to reside primarily in the cytoplasm and RNA-Seq data indicates that approximately 1,400 copies of CDR1as are present in each HEK293 cell. The authors’ data also suggest that CDR1as acts as a miRNA sponge for miR-7 in neuronal tissues and that ectopic expression of human CDR1as in zebrafish causes effects similar to that of knocking down miR-7 expression, supporting the role of CDRA1as as a miRNA sponge.
Taken together these results indicate that circRNAs now comprise an entirely new group of post-transcriptional regulators that can bind miRNA with a capacity greater than any other known type of RNA molecule. These findings also provide another mechanism by which miRNA activity can be regulated.
Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, Maier L, Mackowiak SD, Gregersen LH, Munschauer M, Loewer A, Ziebold U, Landthaler M, Kocks C, le Noble F, & Rajewsky N (2013). Circular RNAs are a large class of animal RNAs with regulatory potency. Nature PMID: 23446348