Circular RNA

Circular RNA (or circRNA) is a type of RNA that, unlike linear RNA, forms a covalently closed continuous loop. In circular RNA the 3' and 5' ends normally present in an RNA molecule have been joined together. This feature confers numerous properties to circular RNAs, many of which have only recently been identified. Though many of these circular RNAs arise from otherwise protein coding genes,^[1] circular RNAs produced in the cell have not been shown to code for proteins. They have therefore been categorized as noncoding RNA. Circular RNAs have recently shown potential as gene regulators. Beyond being a potentially major method of gene regulation that is evolutionarily conserved across species, these molecules may represent new methodologies to treat various diseases, such as cancer and Parkinson’s disease.  

Formation of Circular RNA

RNA Splicing

In order to understand how circular RNAs are formed and why they were once considered "outliers," it is important to briefly outline the process of pre-messenger RNA splicing. In contrast to genes in bacteria, Eukaryotic genes were surprisingly found to be "split." In eukaryotes, as a gene is transcribed from DNA into a primary RNA transcript by RNA polymerase II (Pol II), intervening sequences known as Introns are removed, leaving only exons in the mature Messenger RNA that can subsequently be translated to produce the protein product.^[2] Small nuclear ribonucleoproteins, or snRNPs, consist of small nuclear RNAs (about 150 nucleotides in length) and protein. Multiple snRNPs join with additional proteins to form the spliceosome complex,^[2] which catalyzes splicing in the following manner:

1. The spliceosome recognizes an intron, which is flanked by specific sequences at its 5’ and 3’ ends, known as a donor splice site (or 5’ splice site) and an acceptor splice site (or 3’ splice site), respectively.
2. The 5’ splice site sequence is then subjected to a nucleophilic attack by a downstream sequence called the branch point, resulting in a circular structure called a lariat.
3. The free 5’ exon then attacks the 3’ splice site, joining the two exons and releasing the intron lariat. The intron lariat is subsequently debranched and quickly degraded.^[2]

Alternative Splicing

Many scientists working on the Human Genome Project were shocked to find only 20-25k genes in the human genome. Based on the relative complexity of human systems vs. those of other species (for example, nematodes and mice also have around 20,000 genes) ^[3] about four times that number. How then is human complexity obtained if the number of genes is not increased? Alternative splicing is a partial explanation for this dramatic finding. The same primary RNA transcript can yield different protein products based on which segments are considered "introns" and which are considered "exons" during each splicing event.^[2] One of the most striking examples of alternative splicing is in the Drosophila DSCAM gene. This single gene can give rise to ~30k distinct alternatively spliced isoforms.^[4]

Non-canonical splicing

Exon scrambling

Exon scrambling, also called exon shuffling, describes an event in which exons are spliced in a "non-canonical" (atypical) order. There are three ways in which exon scrambling can occur:

1. Tandem exon duplication in the genome, which often occurs in cancers
2. Trans-splicing (in which two RNA transcripts fuse), which results in a linear transcript that contains exons that, for example, may be derived from genes encoded on two different chromosomes. Trans-splicing is very common in C. elegans
3. A splice donor site being joined to a splice acceptor site further upstream in the primary transcript, yielding a circular transcript.^[1]

The long-held belief that circularized transcripts are accidental byproducts resulting from very rare errors during splicing has recently been challenged.^[1][5][6]

CircRNAs as Viroids

In 1976, HL Sanger published a paper in which he claimed to find the first “single-stranded covalently closed circular RNA molecule”.^[7] This molecule was a viroid. Viroids are not equivalent to viruses.

Sanger’s paper provided evidence for the "true circularity" of viroids. Linear RNAs usually have a free 5’ and 3’ terminus. However, Sanger & group found that they could not phosphorylate the viroid RNA at the 5’ terminus. Then, in another test, they failed to find even one free 3’ end, which ruled out the possibility of the molecule having two 3’ ends. Viroids thus represented truly circular RNAs.

 

Characteristics of Circular RNA

Abundance of circRNAs

At least three groups have addressed the question “How abundant are circular RNAs?” by sorting through vast collections of RNA sequencing data.^[1][6][8] Since the most abundant RNA type in the cell is ribosomal RNA, which is not circular, the groups depleted ribosomal RNA (to generate so-called Ribominus data) prior to generating deep sequencing libraries. To then identify potential circular RNA isoforms, they looked for sequencing reads showing a junction between two “scrambled” exons.

A brief summary of each group’s results follows:

Salzman et al 2012

• Originally wanted to identify cancer-specific exon scrambling events
• Ended up finding scrambled exons in a large number of both normal and cancer cells
• Scrambled exon isoforms: ~10% of total transcript isoforms in leukocytes
• Identified 2,748 scrambled isoforms in HeLa and H9 embryonic stem cells
• About 1 in 50 expressed genes produced scrambled transcript isoforms at least 10% of the time
• Some tests for circularity: (1) Treated samples with RNase R, an enzyme which degrades linear RNAs but not circular RNAs, and (2) Tested for the presence of poly-A tails (shouldn’t be present in a circular molecule)
  • Conclusion – 98% of scrambled isoforms represented circRNAs ^[1]

Jeck et al 2013

• Treated human fibroblast RNA with RNase R to enrich for circular RNAs
• Used three “stringency” categories (low, medium, high) to classify circular transcripts based on their levels of abundance
• Including the “low” category, ~1 in 8 expressed genes produced detectable levels of circRNA
• Significantly higher than Salzman’s number (above)
• May be due to greater sequencing depth ^[6]

Memczak et al 2013

• Studied HEK 293 (Human Embryonic Kidney) cells
• Compared their data to Salzman’s data. Taking overlaps into account, the two groups found 1950 total circRNAs ^[8]

CircRNAs are long

A recent study of human circRNAs revealed that these molecules are usually composed of 1-5 exons.^[8] Each of these exons can be up to 3x longer than the average expressed exon,^[6] suggesting that exon length may play a role in deciding which exons to circularize. 85% of circularized exons overlap with exons that code for protein,^[8] although the circular RNAs themselves do not appear to be translated. During circRNA formation, exon 2 is often the upstream "acceptor" exon.^[1]

Introns surrounding exons that are selected to be circularized are, on average, up to 3x longer than those not flanking pre-circle exons,^[1][6] although it is not yet clear why this is the case. Compared to regions not resulting in circles, these introns are much more likely to contain complementary inverted Alu repeats; Alu is the most common transposon in the genome.^[6] By the Alu repeats base pairing to one another, it has been proposed that this may enable the splice sites to find each other, thus facilitating circularization.^[5][6]

Location of circRNAs in the Cell and Body

In the cell, circRNAs are predominantly found in the cytoplasm, where the number of circular RNA transcripts derived from a gene can be up to ten times greater than the number of associated linear RNAs generated from that locus. This indicates that circRNAs are not accidental errors that are quickly removed from the cell.^[1][5][8] Scientists are somewhat puzzled by the cytoplasmic location of circRNAs. How can circular molecules exit the nucleus through a relatively small nuclear pore? Because the nuclear envelope breaks down during mitosis, one hypothesis is that the molecules exit the nucleus during this phase of the cell cycle.^[6] However, there may be a flaw with this hypothesis: certain circRNAs such as CiRS-7/CDR1as (see below), seem to be important in neuronal tissues,^[8][9] where mitotic division is not prevalent.

In September 2013, it was determined that circular RNA expression is highly specific to cell type and/or developmental stage. This strongly suggests that circRNAs play developmental or regulatory roles in controlling gene expression patterns that distinguish one tissue from another.^[1][8][10] For example, as mentioned above, CiRS-7 / CDR1as is highly expressed in neuronal tissues.^[8][9][10]

Plausible Functions of Circular RNA

Evolutionary Conservation of Circularization Mechanisms & Signals

CircRNAs have been identified in various species across the domains of life. In 2011, Danan et al sequenced RNA from Archaea. After digesting total RNA with RNase R, they were able to identify circular species, indicating that circRNAs are not specific to eukaryotes.^[11] However, these archaeal circular species are probably not made via splicing, suggesting that other mechanisms to generate circular RNA likely exist.

In a closer evolutionary connection, a comparison of RNA from mouse testes vs. RNA from a human cell found 69 orthologous circRNAs. For example, both humans and mice encode the HIPK2 and HIPK3 genes, two paralogous kinases which produce a large amount of circRNA from one particular exon in both species.^[6] Evolutionary conservation reinforces the likelihood of a relevant and significant role for RNA circularization.

CDR1as/CiRS-7 as a miR-7 sponge

microRNAs (miRNAs) are small (~21nt) non-coding RNAs that repress translation of messenger RNAs involved in a large, diverse set of biological processes.^[12] They directly base-pair to target messenger RNAs (mRNAs), and can trigger cleavage of the mRNA depending on the degree of complementarity. MicroRNAs are grouped in “seed families.” Family members share nucleotides #2-7, called the seed region, in common.^[13] Argonaute proteins are the “effector proteins” which help miRNAs carry out their job. MicroRNA sponges do exactly what their name implies. Due to the presence of multiple binding sites that recognize a specific seed region, these RNAs can “sponge up” miRNAs of a particular family, thereby serving as competitive inhibitors that suppress the ability of the miRNA to bind its mRNA targets.^[13] Certain circular RNAs have many miRNA binding sites, which yielded a clue that they may function in sponging. Two recent papers confirmed this hunch by investigating a circular sponge called CDR1as/CiRS-7 in detail.

CDR1as/CiRS-7 is encoded in the genome antisense to the human CDR1 (gene) locus (hence the name CDR1as),^[8] and targets miR-7 (hence the name CiRS-7 – Circular RNA Sponge for miR-7).^[9] It has over 60 miR-7 binding sites, far more than any known linear sponge.^[8][9]

‘‘‘AGO2’’’ is miR-7’s associated Argonaute protein (see above). Though CDR1as/CiRS-7 can be cleaved by miR-671 and its associated Argonaute protein,^[9] it cannot be cleaved by miR-7 and AGO2. MicroRNA cleavage activity depends on complementarity beyond the 12th nucleotide position; none of CiRS-7’s binding sites meet this requirement.

An experiment with zebrafish, which do not have the CDR1 locus in their genome, provides evidence for CiRS-7’s sponge activity. During development, miR-7 is strongly expressed in the zebrafish brain. To silence miR-7 expression in zebrafish, Memczak and colleagues took advantage of a tool called morpholino, which can base pair and sequester target molecules.^[14] Interestingly, morpholino treatment had the same severe effect on midbrain development as ectopically expressing CiRS-7 in zebrafish brains using injected plasmids. This indicates a significant interaction between CiRS-7 and miR-7 in vivo.^[8]

Another notable circular miRNA sponge is SRY. SRY, which is highly expressed in murine testes, functions as a miR-138 sponge.^[9][15] In the genome, SRY is flanked by long inverted repeats (IRs) over 15.5 kilobases (kb) in length. When one or both of the IRs are deleted, circularization does not occur. It was this finding that introduced the idea of inverted repeats enabling circularization.^[16]

Because circular RNA sponges are characterized by high expression levels, stability, and a large number of miRNA binding sites, they are likely to be more effective sponges than those that are linear.^[5]

Other Possible Functions for circRNAs

Though recent attention has been focused on circRNA’s “sponge” functions, scientists are considering several other functional possibilities as well. For example, some areas of the mouse adult hippocampus show expression of CiRS-7 but not miR-7, suggesting that CiRS-7 may have roles that are independent of interacting with the miRNA.^[8]

Potential roles include the following:

• Binding to RNA-binding proteins (RBPs) and RNAs besides miRNAs to form RNA-protein complexes.^[5] These complexes could regulate RBP & RNA interactions with, for example, the canonical linear transcript of the gene.^[1]
• Protein production
  • Chen and Sarnow 1995 showed that a synthetic circRNA that contained an IRES (internal ribosome entry site) produced a protein product ‘‘in vitro’’, whereas that without an IRES did not. Although the tested circRNA was a purely artificial construct, Chen and Sarnow stated in their paper that they would be interested to see whether circles naturally contain IRES elements.^[17]
  • Jeck et al 2013: Tested natural circRNAs that contained a translation "start codon." However, none of these molecules bound to ribosomes, suggesting that many circRNAs may not be translated ‘‘in vivo’’.^[6]
• Transporting miRNAs inside the cell. The fact that CiRS-7 can be sliced by miR-671 might indicate the existence of a system to release a “load” of miRNAs at the appropriate time.^[10]
• Regulating mRNA in the cell through limited base pairing. It is formally possible that miR-7 moderates CiRS-7’s regulatory activity instead of the other way around ^[8][10]!

Circular intronic long non-coding RNAs (ciRNAs)

Usually, intronic lariats (see above) are debranched and rapidly degraded. However, a debranching failure can lead to the formation of circular intronic long non-coding RNAs, also known as ciRNAs.^[18] CiRNA formation, rather than being a random process, seems to depend on the presence of specific elements near the 5’ splice site and the branchpoint site (see above).

CiRNAs are distinct from circRNAs in that they are prominently found in the nucleus rather than the cytoplasm. In addition, these molecules contain few (if any) miRNA binding sites. Instead of acting as sponges, ciRNAs seem to function in regulating the expression of their parent genes. For example, a relatively abundant ciRNA called ci-ankrd52 positively regulates Pol II transcription. Many ciRNAs remain at their “sites of synthesis” in the nucleus. However, ciRNA may have roles other than simply regulating their parent genes, as ciRNAs do localize to additional sites in the nucleus other than their “sites of synthesis”.^[18]

 

Circular RNA and Disease: Pathways for Future Research

As with most topics in molecular biology, it is important to consider how circular RNA can be used as a tool to help mankind. Given its (1) abundance, (2) evolutionary conservation, and (3) potential regulatory role, it is worthwhile to look into how circular RNA can be used to study pathogenesis and devise therapeutic interventions. For example:

• Circular ANRIL (cANRIL) is the circular form of ANRIL, a long non-coding RNA (ncRNA). Expression of cANRIL is correlated with risk for atherosclerosis, a disease in which the arteries become hard. It has been proposed that cANRIL can modify INK4/ARF expression, which, in turn, increases risk for atherosclerosis.^[19] Further study of cANRIL expression could potentially be used to prevent or treat atherosclerosis.

• miR-7 plays an important regulatory role in several cancers and in Parkinson’s disease, which is a degenerative neuronal illness.^[9] Perhaps CiRS-7’s sponge activity could help in countering miR-7 activity. If circular sponge activity can indeed help in countering harmful miRNA activity, scientists will need to figure out the best way to introduce sponge expression, perhaps via a transgene, which is a synthetic gene that is transferred between organisms. It is also important to consider how transgenes can be expressed only in specific tissues, or expressed only when induced.^[13]

External links

• starBase database: a database for exploring miRNA-circRNA interactions from PAR-CLIP(CLIP-Seq, HITS-CLIP,iCLIP,CLASH) data.^[20]
• circBase database: a database for circRNAs.

References