Best practice standards for circular RNA research

Abstract

Circular RNAs (circRNAs) are formed in all domains of life and via different mechanisms. There has been an explosion in the number of circRNA papers in recent years; however, as a relatively young field, circRNA biology has an urgent need for common experimental standards for isolating, analyzing, expressing and depleting circRNAs. Here we propose a set of guidelines for circRNA studies based on the authors' experience. This Perspective will specifically address the major class of circRNAs in Eukarya that are generated by a spliceosome-catalyzed back-splicing event. We hope that the implementation of best practice principles for circRNA research will help move the field forward and allow a better functional understanding of this fascinating group of RNAs.

Figure 1:. Overview of circRNA biogenesis and function

A) Spliceosome-dependent circRNAs are formed by back-splicing of one or more exons that can also undergo linear splicing. The splice sites used to form the circRNA-specific Back-Splice Junction (BSJ) are labelled in yellow (5’SS) and orange (3’SS). B) Nuclear circRNAs have been linked to transcriptional regulation via protein recruitment or as structural components. Cytoplasmic circRNAs are thought to act in a number of ways including as sponges for miRNAs and/or proteins, as scaffolds for complex assembly that may facilitate post-translational modification (e.g., phosphorylation (P), ubiquitylation (Ub) and acetylation (Ac)) and as templates for translation.

Figure 2:. CircRNA detection, validation and quantification

A) RT-PCR-based strategies can detect circular and linear RNAs from the same locus using divergent and convergent primer pairs. Sanger sequencing of the PCR product can confirm BSJ formation (marked in yellow and orange) B) Hybridization-based detection of circRNA by Northern blot. Probes recognize only the circRNA (P1), only the linear RNA (P2) or both forms simultaneously (P3). The inclusion of an RNase R-treated sample can help identify the band that corresponds to the circRNA. C) Direct circRNA detection using nanoString. This technique uses a capture probe and reporter probe that jointly recognize the BSJ to detect individual circRNAs. The amount of reporter probe is quantified using digital counting, thus avoiding an RT-step that may introduce bias. Probe sets can be designed for the circular (1,2) or linear form (3) of a given RNA. D) Splint-Quant is an RT-free technique to quantify circRNAs. The two halves of the BSJ are recognized by donor and acceptor probes that can only be ligated and amplified upon binding to the correct target sequence. E) BaseScope uses Z probes that bind on the two halves of the BSJ. The probes need to bind their target in close proximity to each other in order to form a docking site for a branched DNA strand that can amplify the signal to the levels required for detection.

Figure 3:. Strategies for circRNA depletion

A) Short interfering (si)RNAs designed against the BSJ (yellow and orange) can drive selective cleavage of circRNAs by RNA interference (RNAi), leaving the linearized RNA subject to degradation by exonucleases (RNA decay). A note of caution for RNAi is that a partial overlap with the siRNA can drive miRNA-like repression of the linear form, thus creating off-target effects. B) Guide (g) RNAs targeting the BSJ can be used for selective degradation of the circRNA form via Cas13, while leaving the linear RNA intact. gRNAs require a more extensive sequence overlap with their target than siRNAs and are therefore less prone to off-target effects. C) CircRNA biogenesis can be disrupted by Cas9-dependent genome editing, e.g., by using gRNAs to remove an intron flanking a circularizing exon or delete sequence elements that bring the two splice sites together (e.g., Alu elements and/or RBP sites). In either case, back-splicing is prevented on the edited allele.

Figure 4:. Over-expression of circRNA

A) In vitro formation of circRNAs: The circRNA sequence is transcribed by T7 RNA polymerase using 5’P-GMP to avoid the presence of a triphosphate on the 5’ end of the RNA. The linear RNA is then circularized using T4 RNA ligase and any remaining, unligated RNA is removed by RNase R treatment or gel purification. B) Possible side-effects triggered by transfection of in vitro generated circRNAs: Immune activation: 5’triphosphates, extended dsRNA regions and circRNAs that lack m⁶A marks may be recognized by innate immune receptors in the cytoplasm and trigger a signaling cascade that confounds the biological effect of the introduced circRNA. Reader proteins fail to bind: Endogenous circRNA function may depend on RNA modifications that serve as docking sites for specific reader proteins. CircRNA-binding proteins fail to bind: Endogenous circRNAs may function as circRNPs and depend on proteins that are loaded co-transcriptionally (e.g., the exon-junction complex). C) A common way to over-express circRNAs is by inserting the exon of interest after a strong promoter (CMV) and between a set of inverted repeats (IR) in a plasmid.