Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins

Abstract

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼ 400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3' end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes.

Keywords: Laccase2; PlexA; circRNA; hnRNP; noncoding RNA; pre-mRNA splicing; repetitive element.

Figure 1.

The D. melanogaster laccase2 gene generates a circular RNA. (A) Twenty micrograms of total RNA from DL1 and S2 cells was subjected to Northern blot analysis and probed for Mbl and Laccase2 expression. β-Actin was used as a loading control. (B) The Laccase2 circular RNA was detected with multiple oligonucleotide probes, including one complementary to the backspliced junction (probe 3). (C) The Laccase2 circular RNA is resistant to RNase R digestion. (D) Eleven micrograms of total RNA from adult D. melanogaster tissues was probed for Laccase2 expression. 18S ribosomal RNA was used as a loading control. (E) Exon/intron structure of the D. melanogaster laccase2 locus, highlighting a 1945-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. (F) The 1945-nt region of the Laccase2 pre-mRNA was cloned downstream from the metallothionein promoter to generate the Laccase2 sense plasmid. The regions targeted by Northern oligonucleotide probes are denoted in red. (G) Plasmids containing the laccase2 region in the sense or antisense orientation were transfected into DL1 (left) or S2 (right) cells, and Northern blots were performed. Endogenous Laccase2 circular RNA expression was observed in the mock-treated samples. (*) Concatenated and/or intertwined circular RNA. β-Actin was used as a loading control.

Figure 2.

Base-pairing between intronic repeats facilitates Laccase2 circular RNA production. (A) Numbering scheme for the Laccase2 sense expression plasmid. The minimal sufficient introns that support circularization (450–1245 plasmid) are shown at the bottom. (B,C) Laccase2 expression plasmids containing deletions at their 5′ ends (B) or 3′ ends (C) were transfected into DL1 cells, and CuSO₄ was added for 14 h where indicated. Northern blots were subsequently performed. (D) Mutations in the repeats (denoted in red) were introduced into the Laccase2 nucleotide 450–1245 expression plasmid. mFold was used to calculate hairpin stabilities, assuming a 7-nt linker (AGAAUUA) between the repeats. (E) An EGFP expression plasmid and Laccase2 expression plasmids containing wild-type (WT) or mutant repeats were transfected into DL1 cells. CuSO₄ was then added for 14 h, and Northern blots were performed.

Figure 3.

Multiple hnRNPs and SR proteins regulate Laccase2 circular RNA expression. (A) Drosophila SL2 cells were treated with the indicated dsRNAs for 4 d, and Northern blots were performed to analyze expression of the endogenous mbl and laccase2 loci. Knockdown of the linear Mbl transcript caused depletion of the Mbl circular RNA but had no effect on Laccase2 circular RNA levels. (B) Northern blots were used to examine Laccase2 expression in DL1 cells that had been treated with 4 μg of the indicated dsRNAs for 3 d. Representative blots are shown. (C) Laccase2 circular RNA levels were quantified using ImageQuant from three independent experiments and were normalized to the “no dsRNA” samples. Data are shown as mean ± SD. (D) Northern blots were used to examine Mbl expression in SL2 cells that had been treated with the indicated dsRNAs for 4 d.

Figure 4.

Combinatorial control of circular RNA levels by hnRNPs and SR proteins. DL1 cells were treated with the indicated pairs of dsRNAs (2 μg of each) for 3 d. Northern blots were then used to examine the expression of the endogenous Laccase2 (A) and PlexA (B) circular RNAs. Representative blots are shown. Circular RNA levels were quantified using ImageQuant from three independent experiments and were normalized to the “no dsRNA” samples. Data are shown as mean ± SD.

Figure 5.

The flanking introns dictate whether 3′ end processing is required for exon circularization. (A) Schematics of Drosophila Laccase2 expression plasmids. The complete SV40 poly(A) (pA) signals, which include the AAUAAA sequence, were replaced with dH3 3′ end processing signals, the MALAT1 triple helix, or the HhRz. Whereas the dH3 sense sequence is cleaved by CPSF73, and HhRz self-cleaves, the mMALAT1_3′ ΔmascRNA sequence (nucleotides 6581–6690 of mouse MALAT1) lacks the tRNA-like structure and is unable to be cleaved by RNase P (Wilusz et al. 2012). (B) Laccase2 (left) or EGFP (right) expression plasmids ending in the designated sequences were transfected into DL1 cells. CuSO₄ was added, and Northern blots were then performed. (C) HeLa cells were transfected with mammalian expression plasmids containing laccase2 nucleotides 100–1945 followed by differing 3′-terminal sequences. Unlike in A, the mMALAT1_3′ region (nucleotides 6581–6754 of mouse MALAT1) was inserted, which includes the tRNA-like structure. When present in the sense orientation, the mMALAT1_3′ region is recognized and cleaved by RNase P (Wilusz et al. 2012). (*) A nonspecific band that is also present in mock-treated cells. (D) Schematics of human ZKSCAN1 expression plasmids (Liang and Wilusz 2014). (E) Plasmids containing ZKSCAN1 nucleotides 400–1782 (left) or ZKSCAN1 nucleotides 1–2232 (right) followed by differing 3′-terminal sequences were transfected into HeLa cells, and Northern blots were performed.

Figure 6.

The laccase2 introns facilitate circularization of diverse exons in fly cells. (A) To facilitate the identification of exon sequences that can be circularized in Drosophila, exon 2 of the Laccase2 sense expression plasmid was replaced with an artificial 57-nt exon composed of restriction enzyme sites. (B,C) Segments of laccase2 exon 2 (numbering scheme as in A) were inserted between the KpnI and XmaI sites. DL1 cells were then transfected, CuSO₄ was added, and Northern blots were performed. To avoid detection of the endogenous Laccase2 circular RNA, a probe complementary to the MCS backspliced junction was used. (D) The Laccase2 MCS vector was able to circularize segments of human ZKSCAN1 exons 2 and 3 (numbering scheme from Liang and Wilusz 2014). The ZKSCAN1 nucleotide 627–646 probe (top) detects linear and circular RNAs derived from the plasmid, whereas the circle junction probe (bottom) detects only properly backspliced RNAs. (E,F) Exon 2 of human HIPK3 (E) and the ciRS7 (CDR1as) exon (F) were likewise circularized in DL1 cells when placed between the laccase2 introns.

Figure 7.

Plasmids for efficient circular RNA expression in mammalian cells. (A) To test the ability of the laccase2 exon to circularize in mammalian cells, the 1945-nt region of the Laccase2 pre-mRNA was cloned into pcDNA3.1(+). (Bottom) In the Laccase2 MCS exon vector, exon 2 was replaced with a 63-nt artificial exon. (B) These plasmids were then compared with analogous pcDNA3.1(+) expression plasmids that are based on the human ZKSCAN1 gene. The CircRNA Mini Vector has short Alu repeats flanking the MCS exon, whereas the ZKSCAN1 MCS vector includes intronic sequences from nucleotides 100–2232 of the previously described ZKSCAN1 sense expression plasmid (Liang and Wilusz 2014). (C) Segments of human ZKSCAN1 exons 2 and 3 (numbering scheme from Liang and Wilusz 2014) were inserted into the multicloning sites of the designated plasmids. Plasmids were then transfected into HeLa cells, and Northern blots were performed. The presence of long flanking repeats (in either the ZKSCAN1 MCS vector or the Laccase2 MCS vector) greatly improved circularization efficiency. (D) The ZKSCAN1 and laccase2 introns generated significantly more ciRS7 circular RNA than the previously described ciRS7 expression plasmid (Hansen et al. 2013).