The Output of Protein-Coding Genes Shifts to Circular RNAs When the Pre-mRNA Processing Machinery Is Limiting

Abstract

Many eukaryotic genes generate linear mRNAs and circular RNAs, but it is largely unknown how the ratio of linear to circular RNA is controlled or modulated. Using RNAi screening in Drosophila cells, we identify many core spliceosome and transcription termination factors that control the RNA outputs of reporter and endogenous genes. When spliceosome components were depleted or inhibited pharmacologically, the steady-state levels of circular RNAs increased while expression of their associated linear mRNAs concomitantly decreased. Upon inhibiting RNA polymerase II termination via depletion of the cleavage/polyadenylation machinery, circular RNA levels were similarly increased. This is because readthrough transcripts now extend into downstream genes and are subjected to backsplicing. In total, these results demonstrate that inhibition or slowing of canonical pre-mRNA processing events shifts the steady-state output of protein-coding genes toward circular RNAs. This is in part because nascent RNAs become directed into alternative pathways that lead to circular RNA production.

Keywords: Laccase2; Pladienolide B; SF3b1; backsplicing; circRNA; cleavage/polyadenylation; exon definition; pre-mRNA splicing; spliceosome; transcription termination.

Figure 1. The Laccase2 circular RNA accumulates to high levels due to its long half-life

(A) Schematics of the Hy_pMT Laccase2 Exons 1–3 and Hy_pMT Laccase2 Exon 2 plasmids. Upon induction of the metallothionein promoter (pMT) by addition of CuSO₄, the ~3.6-kb pre-mRNA derived from the Hy_pMT Laccase2 Exons 1–3 plasmid can be canonically spliced to generate a ~1.8-kb linear mRNA that is subsequently polyadenylated (top) or backspliced to generate a 514-nt circular RNA from exon 2 (bottom). Inverted DNAREP1_DM repeat sequences (green) are present in the introns flanking exon 2. See also Figure S1A. (B) Laccase2 plasmids were transfected into Drosophila DL1 cells, CuSO₄ added for 14 h, and Northern blots performed. *Concatenated and/or intertwined circular RNA. β-actin was used as a loading control. See also Figure S1B. (C) A DL1 cell line stably expressing the Hy_pMT Laccase2 Exons 1–3 plasmid was treated with CuSO₄ for the indicated amounts of time, and Northern blots performed. See also Figure S1F. (D) To measure RNA half-lives, the stable cell line was treated with CuSO₄ for 3 h followed by chelation of the metal with BCS for the indicated amounts of time. Cross-hybridization of the Northern probe with ribosomal RNAs is responsible for the ~2-kb transcripts weakly detected after 12 h. See also Figure S1G.

Figure 2. The SF3b and SF3a complexes regulate Laccase2 circular RNA levels

(A and B) A DL1 cell line stably expressing the Hy_pMT Laccase2 Exons 1–3 plasmid was treated with the indicated dsRNAs for 3 d and, where noted, CuSO₄ was added for the last 14 h. Northern blots were used to examine expression of reporter-derived transcripts. Representative blots are shown. See also Figure S2. (C) Linear mRNA and circular RNA levels were quantified using ImageQuant from three independent Northern blot experiments. Data are normalized to the β-gal dsRNA samples and are shown as mean±SD. * p<0.01. (D) DL1 cells were treated with the indicated dsRNAs for 3 d. qRT-PCR was then used to quantify expression of the endogenous Laccase2 linear mRNA (Exon 2/3 junction) and Laccase2 circular RNA. Data from four independent experiments were normalized to β-actin (Act42a) and are shown as mean±SD. * p<0.01. (E) Northern blots were used to measure expression of the endogenous laccase2 gene in DL1 cells that had been treated with dsRNAs for 3 d.

Figure 3. Depleting or pharmacologically inhibiting SF3b results in increased expression of endogenous circular RNAs

(A) DL1 cells were treated with the indicated dsRNAs for 3 d. Northern blots (top) and qRT-PCR (bottom) were then used to quantify expression of the endogenous Uex linear mRNA and circular RNA. A representative blot is shown. Data from four independent qRT-PCR experiments were normalized to β-actin (Act42a) and are shown as mean±SD. * p<0.05. (B) DL1 cells were treated as in A. Northern blots (top) and qRT-PCR (bottom) were used to quantify expression of the endogenous PlexA linear mRNA and circular RNA. A representative blot is shown. Data from four independent qRT-PCR experiments were normalized to β-actin (Act42a) and are shown as mean±SD. * p<0.05. (C) A DL1 cell line stably expressing the Hy_pMT Laccase2 Exons 1–3 plasmid was treated with increasing concentrations of Pladienolide B (Plad B) and, where noted, CuSO₄ for 6 h. Northern blots were used to examine expression of reporter-derived transcripts. (D) DL1 cells were treated with DMSO or 5 nM Plad B for 6 h. Total RNA was then isolated and subjected to qRT-PCR to measure the expression of transcripts derived from the PlexA, Uex, and laccase2 genes. Data from three independent experiments were normalized to β-actin (Act42a) and are shown as mean±SD. * p<0.05.

Figure 4. Depleting many core spliceosomal components results in increased expression of endogenous circular RNAs

(A) DL1 cells were treated with the indicated dsRNAs for 3 d and Northern blots used to quantify expression of the endogenous PlexA linear mRNA and circular RNA. Core spliceosomal components that function at different stages of spliceosome assembly were individually depleted. See also Figure S3. (B) Total RNA was isolated from DL1 cells treated with dsRNAs for 3 d and subjected to qRT-PCR to measure the expression of endogenous circular RNAs. Data from four independent experiments were normalized to β-actin (Act42a) and are shown as mean±SD.

Figure 5. Readthrough transcription from the upstream HygroR gene enables circular RNA production from the minigene reporter

(A) A DL1 cell line stably expressing the Hy_pMT Laccase2 Exons 1–3 plasmid was treated with the indicated dsRNAs for 3 d and, where noted, CuSO₄ was added for the last 14 h. Northern blots were used to examine expression of reporter-derived transcripts. See also Figure S4A. (B) Linear mRNA and circular RNA levels were quantified using ImageQuant from four independent Northern blot experiments and were normalized to the “β-gal, No copper” samples. Data are shown as mean±SD. * p<0.05. (C) Schematic of readthrough transcription model. In control treated cells (top), only the upstream copia transposon LTR promoter is active when no CuSO₄ is present. This results in the production of the HygroR mRNA, which is cleaved/polyadenylated, and the downstream RNA is rapidly degraded by the Rat1 exonuclease to facilitate transcription termination. When Cpsf73 is depleted (bottom), 3′ processing of the HygroR mRNA is inefficient, resulting in readthrough transcription into the downstream laccase2 minigene. When the intronic repeats (green) base pair to one another, a circular RNA is produced. See also Figure S4. (D) DL1 cells stably expressing the Hy_pMT Laccase2 Exons 1–3 plasmid were treated with the indicated dsRNAs for 3 d and total RNA isolated. Northern blots using probes complementary to Laccase2 exon 2 (Lanes 1–4) or the HygroR mRNA (Lanes 5–8) were performed. (E) The laccase2 minigene from the Hy_pMT Laccase2 Exons 1–3 plasmid was replaced with an eGFP open reading frame. In addition, the downstream polyadenylation signal was replaced with the hammerhead ribozyme (HhRz) preceded by a 74-nt sequence from the 3′ end of the Rift Valley Fever Virus (RVFV) NSs mRNA (brown). A DL1 cell line stably expressing this plasmid was then generated and treated with the indicated dsRNAs for 3 d. Depletion of Cpsf73 resulted in the production of a fusion HygroR-eGFP mRNA that is stabilized at its 3′ end by the RVFV NSs sequence.

Figure 6. Readthrough transcription generates endogenous circular RNAs from the human PAIP2 gene

(A) Readthrough transcription from MATR3 as shown by nascent RNA-seq in human PA1 cells (Zhang et al., 2016). The downstream PAIP2 gene generates a circular RNA from exons 2 and 3 (purple). See also Figure S5. (B) Immunoblot to confirm depletion of CPSF3 (top) or CPSF4 (bottom) by shRNAs in PA1 cells. scr, scrambled shRNA control. Actin was used as a loading control. (C) Schematic showing qRT-PCR primers used to quantify transcripts from the MATR3-PAIP2 locus. (D and E) qRT-PCR quantification of transcripts from the MATR3-PAIP2 intergenic region (D) or the PAIP2 gene (E) in PA1 cells depleted of CPSF3 or CPSF4. Data are shown as mean±SD from three independent experiments. * p<0.05, ** p<0.01 (F) PA1 cells were treated for 8 h with a phosphorothioate-modified antisense oligodeoxynucleotide (ASO) complementary to the MATR3-PAIP2 intergenic region. (G) qRT-PCR was then used to quantify transcripts from the MATR3-PAIP2 intergenic region or the PAIP2 gene. Locations of primers are shown in F. Data are shown as mean±SD from three independent experiments. * p<0.05, ** p<0.01

Figure 7. Proposed models for how inhibition or slowing of canonical pre-mRNA processing events can result in increased circular RNA levels

(A) In wild-type (WT) cells (left), exons within pre-mRNAs are first defined and spliceosomal components assemble across each exon. U1 snRNP recognizes the downstream 5′ splice site, U2 snRNP binds the upstream polypyrimidine tract and branch point sequence, and factors such as SR proteins mediate cross-exon interactions. These cross-exon interactions are subsequently replaced with cross-intron interactions to enable full assembly of the spliceosome and generation of a linear mRNA. When spliceosome activity is limiting (e.g. due to depletion of core spliceosome components) (right), we propose that cross-exon interactions are not easily replaced with cross-intron interactions. The full spliceosome thus assembles across an exon, resulting in backsplicing and the generation of a circular RNA. (B) Failure to efficiently terminate transcriptional units (e.g. due to depletion of cleavage/polyadenylation factors) can cause nascent RNAs to be extended into downstream genes. If the appropriate signals are present in this readthrough transcript (such as inverted intronic repeats), backsplicing can occur to release a mature circular RNA. The remaining nascent RNA likely consists of a Y-shaped structure with a 2′-5′ phosphodiester bond at the upstream branch site. This structure may be debranched, thereby providing an entry site for exonucleases, including Rat1, that enable transcription termination.