Engineering and expressing circular RNAs via tRNA splicing

Abstract

Circular (circ)RNAs have recently become a subject of great biologic interest. It is now clear that they represent a diverse and abundant class of RNAs with regulated expression and evolutionarily conserved functions. There are several mechanisms by which RNA circularization can occur in vivo. Here, we focus on the biogenesis of tRNA intronic circular RNAs (tricRNAs) in archaea and animals, and we detail their use as research tools for orthogonal, directed circRNA expression in vivo.

Keywords: Back-splicing; RNA aptamers; circRNA; ectopic gene expression; tRNA processing; tricRNA.

Figure 1.

Two in vivo RNA circularization pathways. (A) Intron base-pairing and/or RNA-binding proteins facilitate pairing of a downstream splice site and an upstream splice site, bringing them into close proximity. Non-canonical “back-splicing” of these sites results in a circularized exon. (B) The tRNA splicing endonuclease (TSEN) complex cleaves an intron-containing pre-tRNA at the bulge-helix-bulge (BHB) motif. The exon halves are ligated, and the intron termini are also ligated to form a circle.

Figure 2.

tricRNA expression can be detected by RT-PCR. (A) Schematic of the tricRNA expression construct, showing the PCR primer binding sites (red arrows) in the intron and an external pol III promoter (blue arrow). Exon A and Exon B represent the sequences present in the mature tRNA. The dotted line indicates a variably-sized intronic region. (B) Reverse transcriptase can transcribe around a tricRNA template many times, resulting in a concatameric cDNA with numerous tandem repeats. (C) RT-PCR primers can bind to multiple sites along the cDNA concatamer, resulting in a ladder of potential PCR products. The formula for the sizes of the bands is: [size of the circle] – [distance between the divergent primers] + [size of circle]n, where n is the number of tandem repeats. (D) The ladder of PCR products can be seen for circles of several different sizes. In this experiment, the distance between the divergent primers is 105 nt, and the sizes of the circles are listed above the gel. For the 259 nt circle, the formula for the ladder is therefore: 259–105+259n. The bands detected on the gel are at 154, 413, 672, and 931 nt.

Figure 3.

Testing in vivo methods of RNA circularization using the Broccoli RNA aptamer as a reporter. (A) Schematic of the 2 constructs, where the bright green box indicates the placement of the Broccoli aptamer sequence. The dotted arcs indicate the splice junctions. The U6* promoter includes the first 27 nucleotides of U6 snRNA in the transcriptional unit. This promotes 5′ capping and enhances stability of the expressed RNA, resulting in a higher yield of tricRNAs. (B) In-gel imaging following transient transfection of the reporter constructs into HeLa and 293T cells. The left hand image is the DFHBI-1T stain, which binds to all Broccoli-containing RNAs. In this image, the top band is the pre-tRNA and the lower band is the circular RNA. The doublet of bands in the first lane is most likely due to transcription beginning from both the external U6 promoter, which has a longer 5′ leader sequence, and the internal tRNA promoter. The right hand image is the ethidium bromide stain of the same gel, which marks total RNA. (C) RT-PCR was performed on cDNA generated from the RNA used in Fig. 3B. Diverging PCR primers were used (similar to Fig. 2A), such that they only generate products of the appropriate size from a circularized template (see also Fig. 2D). In this experiment, the lengths for tricBroc and circBroc are 77 nt and 76 nt, respectively. (D) Northern blot analysis was performed to quantitatively assess circRNA expression in 293T cells.