RNA circularization strategies in vivo and in vitro

Abstract

In the plenitude of naturally occurring RNAs, circular RNAs (circRNAs) and their biological role were underestimated for years. However, circRNAs are ubiquitous in all domains of life, including eukaryotes, archaea, bacteria and viruses, where they can fulfill diverse biological functions. Some of those functions, as for example playing a role in the life cycle of viral and viroid genomes or in the maturation of tRNA genes, have been elucidated; other putative functions still remain elusive. Due to the resistance to exonucleases, circRNAs are promising tools for in vivo application as aptamers, trans-cleaving ribozymes or siRNAs. How are circRNAs generated in vivo and what approaches do exist to produce ring-shaped RNAs in vitro? In this review we illustrate the occurrence and mechanisms of RNA circularization in vivo, survey methods for the generation of circRNA in vitro and provide appropriate protocols.

Figure 1.

Regular linear splicing (a) and two models for the formation of exonic circRNAs (b, c). (a) Upon folding, the branch point adenosine (bpA) attacks the 5′- splice site, delivering the 5′-exon with free 3′-OH group and the lariat intermediate with the intron still linked to the 3′-exon. Nucleophilic attack of the 3′-OH group of the 5′-exon onto the 3′-splice site leads to ligation of the two exons, and to release of the intron as lariat. (b) Direct backsplicing. Two unspliced introns interact by complementary base pairing, thereby juxtaposing the branch point of the 5′-intron and the 3′-intron–exon junction (3′-splice donor) for nucleophilic attack and cleavage. Then, the 3′-splice donor attacks the 5′-intron–exon junction (5′-splice acceptor) joining the two introns and releasing the circularized exon. (c) Exon skipping. Through skipping of an exon, an exon containing lariat is created following the normal mechanism of splicing. Backsplicing then occurs as described above, but within the lariat. As a result, the intron lariat is released and a circular RNA is produced.

Figure 2.

Formation of intronic circRNAs. (a) Group II intron mediated circRNA formation. Circle formation requires prior release of the 3′-exon. The terminal 2′-OH group of the intron attacks the 5′-splice site, creating a circular RNA by 2′,5′-phosphodiester formation. (b) Group I intron supported regular splicing. An exogenous guanosine (exoG) bound in the intron structure serves as nucleophile attacking the 5′-splice site. Upon first transesterification, the 5′-exon is cut off and exoG becomes linked to the intron. The terminal 3′-OH group of the 5′-exon then attacks the 3′-splice site, the ligated exons and a linear intron are released. Eventually the linear intron is circularized by nucleophilic attack of 2′-OH group of the terminal guanosine (ωG) onto a phosphodiester bond close to the 3′-end and release of a short 3′-tail. Note that in this case a 2′-5′-phosphodiester bridge closes the circle. (c) Prior hydrolysis of exon 2 allows circle formation by direct nucleophilic attack of ωG onto the 5′-splice site.

Figure 3.

Double rolling circle mechanism as occurring in viroid-like satellite RNAs (50). Here, self-cleavage of the multimeric (−)-strand is mediated by a hammerhead ribozyme, whereas self-cleavage of the multimeric (+)-strand occurs by hairpin ribozyme action. A similar mechanism is employed in the hepatitis-δ-virus replication, as well as in other satellite RNAs and viroids.

Figure 4.

RNA circularization by chemical ligation with cyanogen bromide in the presence of a morpholino derivative as activator. R = CH₃; CH₂CH₂SO₃H.

Figure 5.

Strategies for juxtaposing reactive ends for enzymatic ligation. Pre-orientation of 5′-p and 3′-OH of the RNA substrate is achieved (a) by hybridization to a splint. This strategy can be used for ligation with T4 DNA ligase (DNA splint required), T4 RNA ligase 1 (RNA splint required) and T4 RNA ligase 2 (DNA or RNA splint) (68); (b) by hairpin and linear helper oligonucleotides (87); (c) by a gap splint that upon hybridization leaves the terminal two or three nucleotides of both ends single stranded at the ligation junction (88); (d) by favorable intrinsic structures (i.e. dumbbell folds (17)). Strategies (b), (d) and (c) are favorable for ligation with T4 RNA ligase 1. All strategies may be used also for chemical RNA ligation. Note that in this case a 3′-phosphate and a 5′-OH group are favorable.

Figure 6.

Comparison of group I intron self-splicing with the permuted intron exon method. (a) Illustration of the circular permutation of the intron. (b) Upon splicing in the normal intron, exons are ligated and the intron is circularized. (c) In the permuted intron, exons are circularized and split introns appear.