Short intronic repeat sequences facilitate circular RNA production

Abstract

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery "backsplices" and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼ 30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3' end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA.

Keywords: Alu; EPHB4; HIPK3; ZKSCAN1; circRNA; noncoding RNA; splicing.

Figure 1.

The human ZKSCAN1 gene generates a circular RNA. (A) Exon/intron structure of the human ZKSCAN1 locus, highlighting a 2232-nt region that includes exons 2 and 3. A circular RNA is formed when the 5′ splice site at the end of exon 3 is joined to the 3′ splice site at the beginning of exon 2 (purple). Repetitive elements in the designated orientations and evolutionary conservation patterns are shown. (B) Ten micrograms of total RNA from 20 normal human tissues was probed for ZKSCAN1 circular RNA expression. 28S ribosomal RNA was used as a loading control. (C) Total RNA from Huh7 cells treated with flavopiridol, a transcriptional elongation inhibitor, was subjected to Northern blot analysis. (D) The 2232-nt region of the ZKSCAN1 pre-mRNA was cloned into pcDNA3.1(+). The regions targeted by Northern oligonucleotide probes are denoted in red. (E) Plasmids containing the ZKSCAN1 region in the sense or antisense orientations were transfected into HeLa cells, and Northern blots were performed. β-Actin was used as a loading control. (F) The ZKSCAN1 circular RNA is resistant to RNase R digestion. (G) Transfected HeLa cells were fractionated to isolate nuclear and cytoplasmic total RNA, which was then subjected to Northern blot analysis with a probe to ZKSCAN1 exon 3. A probe to endogenous MALAT1 was used as a control for fractionation efficiency.

Figure 2.

Short repeat sequences are sufficient to support ZKSCAN1 circularization. (A) Numbering scheme for the ZKSCAN1 expression plasmid. (B,C) ZKSCAN1 plasmids containing deletions at their 5′ ends were transfected into HeLa cells, and Northern blots were performed. The asterisk indicates an additional circular RNA species (see the text). (D) Using ZKSCAN1 expression plasmids starting at nucleotide 400, deletions were similarly introduced into the downstream intron. (E) Sequences of the minimal upstream and downstream introns that support circularization (400–1782 Δ440–500 Δ1449–1735 plasmid) are shown at the bottom and top, respectively. Repeat sequences (green) and splice sites (brown) are highlighted.

Figure 3.

Only specific short repeat sequences are able to support circular RNA production. (A) Mutations (denoted in red) in the minimal sufficient repeat sequences were introduced into the ZKSCAN1 expression plasmid. mFold was used to calculate hairpin stabilities, assuming a 7-nt linker (AGAAUUA) between the two repeat sequences. (B,C) ZKSCAN1 plasmids containing wild-type (WT) or mutant repeats were transfected into HeLa cells, and Northern blots were performed. The minimal sufficient introns (as depicted in Fig. 2E) were used in B, while slightly longer introns were used in C. (D) Although sufficient for circular RNA production, the minimally sufficient AluSq2 region (nucleotides 400–439) was not required for circularization. (E) The minimal AluSq2/AluSz repeats (nucleotides 400–439 and 1747–1782) were replaced with other 40-nt regions of the Alu elements. The remainder of the plasmid was unchanged, allowing the effect of altering only the repeat sequences to be measured. mFold was used to calculate hairpin stabilities as above. (F) Northern blots revealed that the thermodynamic stability of the hairpins is not an adequate predictor of circularization efficiency.

Figure 4.

Specific short repeats are sufficient for HIPK3 circularization. (A) Exon/intron structure of the human HIPK3 locus, highlighting a 2803-nt region that includes exon 2. A circular RNA is formed when the 5′ splice site at the end of exon 2 is joined to the 3′ splice site at the beginning of exon 2 (purple). Repetitive elements in the designated orientations are shown. (B) Ten micrograms of total RNA from 20 normal human tissues was probed for HIPK3 circular RNA expression. 28S ribosomal RNA was used as a loading control. (C) Nucleotides 300–331 of the upstream AluSz element are highly complementary to two different regions of the downstream AluSq2 element. Sequence differences between the two downstream elements are shown in red. (D) Deletions were introduced into the HIPK3 expression plasmid. The two AluSq2 complementary regions are highlighted in yellow. (E) HIPK3 plasmids were transfected into HeLa cells, and Northern blots were performed. Deleting portions of the 2607–2638 complementary region eliminated circular RNA production.

Figure 5.

EPHB4 circularization is inhibited by a portion of the flanking Alu repeats. (A) Exon/intron structure of the human EPHB4 locus, highlighting a 1428-nt region that includes exons 11 and 12. A circular RNA is formed when the 5′ splice site at the end of exon 12 is joined to the 3′ splice site at the beginning of exon 11 (purple). Repetitive elements in the designated orientations are shown. (B) Schematics of EPHB4 expression plasmids. Exon 12 and the downstream intron are not shown for simplicity. (C) EPHB4 plasmids were transfected into HeLa cells, and Northern blots were performed. Probe-binding sites are shown in Supplemental Figure 7C. The 250–1428 plasmid contains 94 nt of the AluSx1 element, but no circularization was observed. In contrast, the 200–1428 Δ251–343 plasmid contains only 50 nt of the AluSx1 element and efficiently generated the EPHB4 circular RNA. (D) The poly(A) tract at the 3′ end of the AluSx1 element (nucleotides 321–343) inhibits circularization.

Figure 6.

3′ end formation is required for circularization. (A) Schematics of ZKSCAN1 expression plasmids. The complete polyadenylation (pA) signals, which include the AAUAAA sequence, were placed in the antisense orientation or replaced by nucleotides 6581–6754 of mouse MALAT1. This 174-nt region, denoted as mMALAT1_3′, includes a tRNA-like structure that is recognized and cleaved by RNase P (Wilusz et al. 2008). U-rich (denoted in red) and A-rich motifs then form a triple helical structure, thereby protecting the mature 3′ end from exonucleolytic degradation (Wilusz et al. 2012). To generate the mMALAT1_3′ Mut U1/U2 plasmid, U → A mutations were introduced into both U-rich motifs, thereby disrupting the triple helix. (B) HeLa cells were transfected with pCRII-TOPO plasmids expressing ZKSCAN1 followed by differing 3′-terminal sequences. Northern blots were then performed using probe 3 from Figure 1D.

Figure 7.

Collaboration between the exon and introns allows circularization. (A) To facilitate the identification of exon sequences that can be circularized, exons 2 and 3 of the minimal ZKSCAN1 400–1782 Δ440–500 Δ1449–1735 expression plasmid were replaced with an artificial 51-nt exon composed of restriction enzyme sites. (B) Segments of the ZKSCAN1 exons were then inserted between the EcoRV and SacII sites. The numbering scheme is as in A and Figure 2A. The intron between exons 2 and 3 (nucleotides 1062–1282) was not included in the vectors designated “No Intron.” HeLa cells were then transfected, and Northern blots were performed. (C) Exons 11 and 12 of EPHB4 with or without the internal intron were inserted into the CircRNA Mini Vector. The numbering scheme is as per Figure 5A. (D) Exon 7 of GAPDH, which is 336 nt, was inserted into the CircRNA Mini Vector. In addition to the circular RNA, the endogenous GAPDH transcript was detected on the Northern blot. (E) The minimal ZKSCAN1 introns were unable to support circularization of HIKP3 exon 2. (F) Comparison of different pre-mRNA splicing mechanisms. Whereas canonical splicing produces a linear mRNA, base-pairing between intronic repeat sequences can trigger backsplicing and the formation of a circular RNA. When base-pairing occurs between complementary sequences on two independent primary transcripts, trans-splicing can generate a chimeric linear mRNA.