Biochemical methods for circRNA characterization

Biochemical experimental methods are important tools for validating and identifying circRNAs. Among the most basic tools to validate a circRNA is reverse transcription-PCR (RT-PCR). In this approach, the circRNA is converted to a cDNA via reverse transcription (RT). Because the cDNA is derived from a circRNA, the sequence contains a diagnostic exon-exon junctional sequence that is absent in the canonically spliced mRNA, and primers can thus be designed to specifically amplify and detect this diagnostic junction. These primers are known as ‘inverse’ or ‘outward-facing’ primers because, when aligned to the genome, their 3′ ends face away from each other, unlike in standard PCR. This prevents the amplification of species that do not contain the diagnostic junction, such as mRNA or genomic DNA. Quantitative RT-PCR (qRT-PCR) can be used to assess quickly the relative abundance of the circRNA across a panel of samples.

Although RT-PCR is very simple and powerful, it also has biases and artifacts. Template switching during the RT step (Fig. 2A) can lead to a variety of artifactual templates that could give rise to PCR products even with inverse primers (Cocquet et al., 2006; Luo and Taylor, 1990; Roy et al., 2015). Thus, it is imperative that the PCR products are sequenced to verify the diagnostic scrambled junction. Even after this verification, it is possible that template switching (Kulpa et al., 1997), trans-splicing (splicing between two separate pre-mRNA molecules) (Agabian, 1990), or unexpected genomic duplication events might have produced a product precisely at the junctional boundary, thus yielding a linear transcript with the scrambled junctional sequence (Fig. 2A). Furthermore, as circRNAs have no defined end, it should be noted that strand-displacing RT enzymes can create long cDNAs consisting of concatemers of exons (Fig. 2B). This phenomenon, known as rolling-circle RT, can lead to a laddering appearance on a gel after PCR (Barrett et al., 2015; You et al., 2015). This can be strong evidence for circularization but can also lead to an overestimation of circRNA by qPCR and sometimes by RNA-seq, depending on the protocol used for library preparation.

Open in a separate window

Fig. 2.

circRNA detection: sources of backsplice junctional sequences. Backsplice junctional sequences can arise from a variety of sources, resulting in false-positive detection and improper quantification of circRNAs. (A) Reverse transcriptase (RT) template switching, trans-splicing, and genomic duplication events can all give rise to linear products with spurious backsplice junctional sequences. These products can be amplified by PCR but are not derived from circRNAs. (B) Rolling-circle RT occurs when a short circRNA is converted to a long cDNA, leading to a concatamer of PCR-amplifiable sequences. Because the primers can bind to this concatemeric sequence in several locations, many PCR products can be created from a single cDNA template.

Northern blotting is also a simple and effective way to detect circRNA (Capel et al., 1993). Probes are designed to target the circularized exonic sequence and can even specifically target the diagnostic junctional sequence. To ensure specificity is not compromised, multiple probes targeting the circRNA can be used in separate blots. Northern blots have the advantage that the species being monitored is known precisely, as it has a particular mobility, whereas in PCR the monitored species need only contain the diagnostic sequence, which may arise from one or more sources of RNA or DNA (Fig. 2A).

RNase R, an exoribonuclease capable of processively degrading RNA from its 3′ to 5′ end, is a useful tool, often used in combination with those described above, to assess whether RNA is indeed circular and to enrich for circRNA in sequencing libraries (Suzuki et al., 2006; Vincent and Deutscher, 2006). However, the use of RNase R can introduce technical noise, as it can decay some circles, perhaps owing to endogenous nicking of the RNA or contaminating nucleases, and some linear molecules are resistant to digestion if their 3′ ends are involved in base pairing (Vincent and Deutscher, 2006). Also, because this enzyme is not completely efficient, some fraction of linear RNA will remain even after treatment. For this reason, it is important to use a quantitative metric such as qRT-PCR to assess its efficacy, as other methods such as endpoint PCR might lead to signal saturation even after RNase treatment. A much less frequently used enzyme, XRN1, a 5′-to-3′ exoribonuclease, can be used in a similar manner as RNAse R, but mRNA must be decapped with tobacco acid pyrophosphatase (TAP) prior to treatment. Despite the imperfections described above, exonuclease resistance along with quantitative measures of resistance to the nuclease (e.g. qRT-PCR or northern blot) provides compelling evidence for circularity.

Another strong assay for circularity involves RNase H, an endoribonuclease that can cleave RNA at RNA-DNA hybrids. In this assay, two short DNA probes are annealed to the RNA of interest, and the RNA is cleaved by RNase H in both locations. When the RNA is run on a gel and blotted for the fragments, two bands should be observed if the RNA is circular or three if the RNA is linear (Capel et al., 1993). When RNA is cleaved with RNase H in the presence of each DNA probe separately, one band will be observed for a circular species and two for a linear. In addition, a change in migration can be observed for the circular species after it is linearized by a single cleavage event (Starke et al., 2015) because linear and circular species do not exhibit the same migration through a polyacrylamide gel matrix.

Two-dimensional denaturing polyacrylamide gel electrophoresis can also be used to distinguish between circRNAs and linear RNAs. In this technique, total RNA is run on a 2D gel containing different percentages of polyacrylamide in each dimension. Linear RNA migrates along the diagonal of the 2D gel according to its size, whereas circRNA travels in an arc owing to its anomalous migration (Tabak et al., 1988). This arc can be excised and sequenced using next generation sequencing (NGS) as an enrichment step (Awan et al., 2013). Alternatively, the 2D gel can be probed (via northern blotting) to quantify or identify particular circRNAs.

In addition to exoribonuclease treatment and 2D gels, ribosomal RNA (rRNA) depletion and polyA-depletion are common methods used to enrich for circRNAs in sequencing libraries. Neither guarantees that the enriched sequences are exclusively circular, as many types of non-coding RNA will also survive these selections. Nonetheless, these biochemical methods can be combined with RNA-seq to assess genome-wide circRNA expression and, in principle, to distinguish artifactual circRNA expression from truly expressed circRNA. For example, intuitively, a comparison of circRNA detection rates in RNase R-treated samples versus ribosomal-depleted samples should provide evidence for the detection of a true circRNA. If such analyses are performed, it is important to normalize appropriately the RNA-seq datasets being compared (Salzman et al., 2011). However, biochemical treatment/purification before RNA-seq does not serve as a sufficient gold standard for genome-wide circRNA identification. For example, Jeck et al. reported depletion of the well-known circle derived from the CDR1 antisense transcript after RNase R treatment (Jeck et al., 2013).

Ectopic expression of circRNAs: gaining insights into function and biogenesis

No methods currently exist to interrupt or induce expression of a specific circRNA from its endogenous locus. Thus, the function (Li et al., 2015; Memczak et al., 2013) and biogenesis (Ashwal-Fluss et al., 2014; Barrett et al., 2015; Kramer et al., 2015; Liang and Wilusz, 2014; Starke et al., 2015) of circRNAs is often studied using easily manipulable circRNA overexpression plasmids. In some cases, entire genes can be expressed on a plasmid (Barrett et al., 2015), although this is often not possible for mammalian genes. To circumvent this issue, mammalian vectors normally only contain the circularized exon(s) along with flanking splicing signals and intronic sequences, which harbor inverted repeats to facilitate their splicing into a circle (Fig. 3A) (Ashwal-Fluss et al., 2014; Hansen et al., 2013; Kramer et al., 2015; Li et al., 2015; Liang and Wilusz, 2014; Starke et al., 2015).

Open in a separate window

Fig. 3.

circRNA overexpression vectors. circRNA overexpression vectors are often used as convenient tools for studying the function and biogenesis of circRNAs. (A) circRNAs are commonly overexpressed on plasmids using gene fragments under the control of a strong promoter (e.g. CMV in mammalian cell lines). Circularization is induced by inverted repeats (IR) flanking the circularized exon, which presumably bring the splice acceptor (SA) and splice donor (SD) into close proximity for backsplicing. (B) In some cases, RNA polymerase (RNA POL) can bypass termination signals, resulting in rolling-circle plasmid transcription. This can give rise to a linear transcript capable of being canonically spliced, yielding a spurious backsplice junctional sequence. TSS, transcription start site.

However, a common technical artifact arising from the use of these vectors occurs as a result of rolling circle transcription of the plasmid (Fig. 3B). For illustration, suppose the plasmid contains the circularized exon of a gene with flanking introns. If the transcription termination signals in the vector are bypassed, the RNA polymerase will continue to transcribe around the entire plasmid, generating a concatamer of the RNA sequence contained in the plasmid. This results in a number of undesired transcripts, including a pre-mRNA containing tandem repeats of the circularized exon, which can then be spliced canonically, pairing upstream donors to downstream acceptors, and yielding a linear concatamer of the circularized exon. Importantly, this RNA will contain a scrambled junction and might appear identical to a bona fide circRNA depending on the experimental approach used for its detection. These artifactual products can lead to off-target effects on the cell and spurious circRNA quantification, especially by qRT-PCR. In some cases, care has been taken in vector design to minimize the expression of erroneous products (Kramer et al., 2015). However, for some applications, such as establishing translation or function of a circRNA, these artifacts must be completely eliminated.

Cell-type and tissue specificity of circRNA expression

A number of circRNAs are expressed in a tissue-dependent manner. In the case of the DCC gene, researchers found that the expression of the circular isoform varied across a variety of human tissues and was not correlated with the expression of its cognate linear mRNA; in some cases, no circRNA was detected despite high levels of mRNA expression (Nigro et al., 1991). Recent studies report similar findings (Rybak-Wolf et al., 2015; Salzman et al., 2013; Szabo et al., 2015; You et al., 2015), demonstrating that this apparent regulation of circularization is a widespread phenomenon. Other studies also demonstrate that global levels of circRNA and mRNA do not correlate and that the diversity of circular isoforms from a particular gene can vary across a panel of cell types (Salzman et al., 2013). Several groups have reported generally high levels of circRNA in the brains of pigs, humans and mice, with especially high levels in the cortex and cerebellum (Rybak-Wolf et al., 2015; Szabo et al., 2015; Venø et al., 2015; You et al., 2015). Furthermore, circRNAs expressed in fly heads or mouse brains are enriched in genes that code for neuronal proteins and synaptic factors, suggesting a potential role for circRNA in the central nervous system (Westholm et al., 2014; You et al., 2015).

The apparent regulation of circRNAs appears to be a general phenomenon that is conserved; even in fission yeast, some circRNAs exhibit changes in abundance that are independent of their linear isoform during nitrogen starvation (Wang et al., 2014). Together, these results strongly suggest that the expression of circRNAs is a regulated process.

The conservation of circRNA expression

circRNA expression across mammals also appears to be conserved. That is, the expression of circRNA isoforms from orthologous genes in mammals is greater than expected by chance. Several studies of circRNA conservation have focused on comparing circRNA expression in mice and humans. Estimates of the fraction of mouse circRNAs with human orthologs range widely from less than 5% to nearly 30% (Guo et al., 2014; Jeck et al., 2013; Memczak et al., 2013; Rybak-Wolf et al., 2015). A recent study of circRNAs in the porcine brain calculated that ~15-20% of the circRNAs produced in the mouse brain use splice sites that are orthologous to those used by circRNAs in the pig brain (Venø et al., 2015). Moderate conservation between pig and human brain circRNAs was also reported, with 5-10% of human brain circRNAs also being expressed in the pig brain; increased rates of conservation (~15%) emerged when considering only a subset of highly expressed circles. Another study estimated 23% conservation between mouse and rat circRNAs, and reported high conservation of circRNAs at the sequence level surrounding the circRNA scrambled junction compared with other splice site-proximal exonic sequences in the host gene (You et al., 2015).

The large variation between these results probably reflects differences in sequencing depth (as many circles have low expression and may escape detection in samples with low coverage), differences in the statistical definition of conservation, bioinformatic parameters used to analyze the datasets, and, importantly, differences in sources of tissue used for comparison. For example, studies determining rates of circRNA conservation between mouse and human samples stratified by tissue type and tested separately will, not surprisingly, yield different results than those performing the same test after collapsing across all organs (i.e. tests of conservation between any cataloged human or mouse sample). This effect is well-known in statistics and is coined Simpson's paradox.

In addition to their conservation across mammals, circRNAs have been, to our knowledge, detected in every eukaryotic species tested to date (Wang et al., 2014). Notably, circRNAs are expressed in microbial eukaryotes including Schizosaccharomyces pombe and Saccharomyces cerevisiae, two highly diverged yeasts with very limited examples of alternative splicing. CircRNA has also been detected in plants (Arabidopsis thaliana), protists (Plasmodium falciparum and Dictyostelium dictostelium) and numerous animals from fly to human (Memczak et al., 2013; Salzman et al., 2013; Wang et al., 2014). These findings suggest that either circRNA is an ancient feature of gene expression, or it has independently arisen several times over the course of eukaryotic evolution.

The dynamic expression of circRNAs during development

circRNAs exhibit dynamic global changes in their expression levels during development. Studies in humans and flies, for example, have found a general induction in circRNA expression during embryonic development (Szabo et al., 2015; Westholm et al., 2014). In humans, induction was observed across a variety of tissues and was consistently observed for circRNAs spliced by both the major (U2) and minor (U12) spliceosome (Szabo et al., 2015). An interesting developmentally regulated circRNA is the conserved circRNA derived from the second exon of NCX1 (SLC8A1 – Human Gene Nomenclature Database). In humans, this circRNA has the highest level of expression and induction of any circRNA expressed during human fetal development. Expressed primarily in the heart, the NCX1 gene encodes a sodium/calcium exchanger responsible for transporting calcium out of the cardiomyocyte after contraction (Jordan et al., 2010). The developmental induction of NCX1 circRNA expression was also recapitulated in vitro with experiments differentiating human embryonic stem cells to cardiomyocytes over the course of 2 weeks (Szabo et al., 2015). A putative function has yet to be ascribed to the NCX1 circRNA isoform.

Many studies have focused specifically on neuronal development because of the high levels of circRNA expression in the brains of flies, mice, pigs and humans (Szabo et al., 2015; Venø et al., 2015; Westholm et al., 2014; You et al., 2015). In the most detailed study to date, You et al. found that circRNAs expressed in mouse brains were enriched in synapses compared with whole hippocampal homogenate and were largely derived from genes associated with synaptic function (You et al., 2015). Analysis of mouse brains from embryonic day (E) 18 to postnatal day (P) 30 revealed that the largest changes in circRNA abundance, both increases and decreases, occurred near the time of synapse formation (P10). Moreover, RNA fluorescence in situ hybridization (FISH) of circRNAs demonstrated localization to the cell body and dendrite, much like mRNAs (Cajigas et al., 2012) and regulatory RNAs such as microRNAs (miRNAs) (Tai and Schuman, 2006). A similar study focusing on pig embryonic brain development found peak circRNA expression at E60, which corresponds to a period of development with high levels of neurogenesis (Venø et al., 2015). Moreover, the circRNAs upregulated at this time point were associated with genes involved in Wnt signaling, axon guidance and TGFβ signaling. In flies, circRNAs were found to accumulate with age in the head and were also highly enriched in genes relating to development and signaling, neurogenesis, and neuronal morphology and function (Westholm et al., 2014).

In vitro experiments using both mouse and human cell lines (P19 cells and SH-SY-5Y cells, respectively) showed global increases in circRNA expression after differentiation to neurons using retinoic acid (Rybak-Wolf et al., 2015). Many circles increased with linear mRNA expression but rarely by the same factor, and some circRNAs even showed an inverse relationship with their cognate linear isoform, although how these changes relate to circRNA function during development requires further investigation.

Two key examples of highly expressed, neural-specific and conserved circRNAs that exhibit developmentally regulated expression are the circRNAs derived from the RIMS2 gene (circRIMS2) and the circRNA derived from the CDR1 antisense locus (ciRS-7) (Rybak-Wolf et al., 2015; Venø et al., 2015). These circRNAs are essentially exclusively expressed in the brain and are conserved across mouse, human and pig. Both circRNAs exhibit a general monotonic induction of expression during neuronal development in vitro (Rybak-Wolf et al., 2015), whereas they appear to exhibit peaks in expression during fetal development in pig (Venø et al., 2015).

The function of specific circRNAs in development

Almost a decade ago, the Drosophila muscleblind (mbl) gene was found to give rise to developmentally regulated, highly abundant transcriptional products now known to be circRNAs (Ashwal-Fluss et al., 2014; Houseley et al., 2006). Because mbl is an essential gene in Drosophila and human (Artero et al., 1998; Begemann et al., 1997), future studies may reveal if and how the circular isoform contributes to this essentiality or plays a role in development. As mentioned above, circMbl might exert these effects by tuning the expression of the mbl transcript, competing with the production of mRNA and binding to MBL protein.

CircRNA from the mouse Sry gene is another early key example of developmentally regulated circRNA splicing. In this case, the linear Sry isoform is expressed in the developing genital ridge where it plays a fundamental role as a transcription factor in sex determination, whereas the circular isoform is expressed in adult testes (Capel et al., 1993). The circularization of the Sry transcript is dictated by promoter usage: the use of a promoter proximal to the coding region gives rise to a linear translated transcript, whereas the use of a distal promoter gives rise to a linear RNA containing long inverted repeats that is spliced to form a circRNA (Fig. 6) (Hacker et al., 1995). These complementary sequences are required for splicing the Sry circRNA (Dubin et al., 1995). Indeed, complementary sequence-mediated circularization appears to be a more general phenomenon; it has been corroborated for several circRNAs in recent years and is now the basis of many circRNA overexpression vectors (Kramer et al., 2015; Liang and Wilusz, 2014; Zhang et al., 2014). Although no function could be ascribed to the Sry circRNA at the time of its discovery, there is now evidence that it might function as a miRNA sponge for miR-138, binding up to 16 molecules of this miRNA per circRNA (Fig. 6; Fig. 7A) (Hansen et al., 2013).

Open in a separate window

Fig. 6.

Developmentally regulated expression of SRY. (A) In the genital ridge of the developing mouse embryo, the Sry transcription start site (TSS) occurs proximal to the open reading frame (ORF), yielding a translatable mRNA that gives rise to SRY protein, a transcription factor involved in sex determination. (B) In the adult testis, the TSS occurs far upstream, yielding a long transcript containing large inverted repeats (green arrows). This transcript is backspliced to form a circRNA, which might function as a miR-138 sponge. SA, splice acceptor; SD, splice donor.

Open in a separate window

Fig. 7.

circRNA functional classes. (A) circRNAs can function as a sponge containing several binding sites for a particular miRNA/RBP and can compete a miRNA/RBP away (dashed arrows) from its mRNA targets, altering gene expression. (B) Through an interaction with U1 snRNP, exon-intron circRNAs (EIciRNAs) can interact with transcription complexes at host genes to induce their transcription (adapted from Li et al., 2015).

Another circRNA named ciRS-7, or sometimes simply CDR1as, which is derived from the Cdr1 antisense locus, also probably functions as a miRNA sponge (Hansen et al., 2013; Memczak et al., 2013). ciRS-7 is very highly expressed in the mammalian brain, is induced during neuronal development (Rybak-Wolf et al., 2015) and has >70 potential miRNA-binding sites for miR-7, most of which are conserved across eutherian mammals (Hansen et al., 2013; Memczak et al., 2013). When ciRS-7 is ectopically expressed in zebrafish, which normally do not express this circRNA but do express miR-7, defects in midbrain development are observed (Memczak et al., 2013), suggesting that this RNA might play a role in the development of the mammalian brain where ciRS-7 and miR-7 are co-expressed (Hansen et al., 2013).

We would like to acknowledge the members of the Salzman lab and the Stanford Biochemistry Department for useful discussions as we prepared this manuscript.