Best practices to ensure robust investigation of circular RNAs: pitfalls and tips

Abstract

Pre-mRNAs from thousands of eukaryotic genes can be non-canonically spliced to generate circular RNAs (circRNAs) that have covalently linked ends. Most mature circular RNAs are expressed at low levels, but some have known physiological functions and/or accumulate to higher levels than their associated linear mRNAs. These observations have sparked great interest into this class of previously underappreciated RNAs and prompted the development of new experimental approaches to study them, especially methods to measure or modulate circular RNA expression levels. Nonetheless, each of these approaches has caveats and potential pitfalls that must be controlled for when designing experiments and interpreting results. Here, we provide practical advice, tips, and suggested guidelines for performing robust identification, validation, and functional characterization of circular RNAs. Beyond promoting rigor and reproducibility, these suggestions should help bring clarity to the field, especially how circular RNAs function and whether these transcripts may sponge microRNAs/proteins or serve as templates for translation.

Keywords: RNase R; backsplicing; circRNA; microRNA sponge; translation.

Figure 1

Annotation of circular RNAs using RNA‐seq reads that span backsplicing junctions. (A) Splice sites can be joined in a linear order by the pre‐mRNA splicing machinery to generate a canonical linear mRNA that is also capped and polyadenylated (top). Alternatively, a pre‐mRNA can be subjected to backsplicing, which yields a circular RNA whose ends are covalently linked (bottom). (B) High‐throughput RNA‐seq reads can be used to annotate RNA isoforms generated from a gene locus. In this example, red sequencing reads do not span a splicing junction and cannot distinguish linear vs. circular RNA isoforms, whereas the green reads are consistent with production of the canonically spliced linear mRNA. The orange read suggests the existence of a transcript that has the end of exon 2 joined to the beginning of exon 2. This is consistent with a backsplicing event that results in production of a circular RNA from exon 2 (as in (A)). (C–E) However, the orange sequencing read may not be derived from a circular RNA backsplicing junction and thus may represent a false positive. (C) If exon 2 is duplicated in the genomic DNA, transcription of the gene results in a linear RNA that has the apparent backsplicing junction. (D) If pre‐mRNAs derived from the gene are subjected to a trans‐splicing event, a linear RNA that has the apparent backsplicing junction is produced. (E) During cDNA synthesis, reverse transcriptase (RT) can dissociate from a template RNA and resume extension from a second template, which can also result in a false‐positive backsplicing junction.

Figure 2

RNA‐seq reads that span backsplicing junctions often do not reveal the internal structure of the circular RNA An example RNA‐seq read (orange) that connects the end of exon 4 to the beginning of exon 2. This may be derived from a circular RNA that contains the three annotated exons or from a circular RNA that has been alternatively spliced. Follow‐up analyses, e.g., using RT–PCR or Northern blots, are required to determine which circular RNA isoform(s) are, in fact, present in cells.

Figure 3

Differences in the sets of transcripts digested by RNase R depend on the assay conditions RNase R digestions are typically performed in a K⁺ containing buffer, but this approach fails to remove many linear RNAs that contain G‐quadruplexes (G4) or have highly structured 3′ ends. By treating purified RNA samples with poly(A) polymerase followed by RNase R digestion in the presence of a Li⁺ containing buffer, linear RNAs are more efficiently removed. This allows circular RNAs to be more highly enriched.

Figure 4

RT–PCR and Northern blotting to validate circular RNA expression (A) By designing different primer pairs, RT–PCR can be used to quantify expression of the linear mRNA (green primers), the circular RNA (orange primers), or both transcripts (purple primers) derived from a gene. (B) Likewise, antisense oligonucleotides that are complementary to the middle of the exon (purple probe) or the backsplicing junction (orange probe) can be used as probes to visualize gene outputs using a Northern blot.

Figure 5

Over‐expression constructs can generate circular RNAs as well as other transcripts Backsplicing can be induced when inverted repeats (gray arrows) in the flanking introns base pair to one another. To generate circular RNA over‐expression constructs, the exon that circularizes along with the immediate flanking sequences is often cloned. This can result in backsplicing (orange) and the production of the circular RNA, but a number of linear transcripts that are not desired are also often produced from these vectors.

Figure 6

Stoichiometry dictates whether a circular RNA can function as a microRNA sponge (Left) A circular RNA that is expressed at low levels and has a single microRNA recognition element (MRE) is unlikely to out‐compete other cellular transcripts for binding to that microRNA. (Right) Only when a circular RNA is highly expressed and has many MREs, does it have the potential to titrate away the microRNA and serve as a sponge.