Use of circular RNAs as markers of readthrough transcription to identify factors regulating cleavage/polyadenylation events

Abstract

Circular RNAs with covalently linked ends are generated from many eukaryotic protein-coding genes when the pre-mRNA splicing machinery backsplices. These mature transcripts are resistant to digestion by exonucleases and typically have much longer half-lives than their associated linear mRNAs. Circular RNAs thus have great promise as sensitive biomarkers, including for detection of transcriptional activity. Here, we show that circular RNAs can serve as markers of readthrough transcription events in Drosophila and human cells, thereby revealing mechanistic insights into RNA polymerase II transcription termination as well as pre-mRNA 3' end processing. We describe methods that take advantage of plasmids that generate a circular RNA when an upstream polyadenylation signal fails to be used and/or RNA polymerase II fails to terminate. As a proof-of-principle, we show that RNAi-mediated depletion of well-established transcription termination factors, including the RNA endonuclease Cpsf73, results in increased circular RNA output from these plasmids in Drosophila and human cells. This method is generalizable as a circular RNA can be easily encoded downstream of any genomic region of interest. Circular RNA biomarkers, therefore, have great promise for identifying novel cellular factors and conditions that impact transcription termination processes.

Keywords: Backsplicing; Cpsf73; RNAi screening; Transcription termination; circRNA; pre-mRNA 3′ end processing.

Fig. 1.. A circular RNA-based reporter plasmid for studying readthrough transcription.

(A) Schematic of the Hy_pMT Laccase2 Exons 1–3 plasmid. The length of each indicated region is denoted in purple. (B) When Drosophila cells are grown under standard conditions (no exogenous copper added to the cell culture medium), the Copia LTR promoter is active, resulting in production of mature (capped and polyadenylated) HygroR mRNAs. The pMT promoter is off and no transcripts are produced from the laccase2 minigene. (C) When exogenous copper is added, the inducible pMT promoter becomes activated and the transcribed pre-mRNAs can be alternatively spliced to yield a three-exon linear mRNA or a circular RNA from exon 2 from the laccase2 minigene. (D) If the SV40 polyadenylation signal located downstream of the HygroR open reading frame fails to be used, there is readthrough transcription into the downstream laccase2 minigene. This can result in production of the circular RNA even though no exogenous copper has been added to cells.

Fig. 2.. Use of a circular RNA-based reporter to identify factors required for transcription termination in Drosophila cells.

(A) A Drosophila DL1 cell line stably maintaining 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. Components of the CPSF, CstF, CF I_m, and CF II_m complexes are indicated. Total RNA was isolated and Northern blots were used to examine expression of transcripts derived from the laccase2 minigene. β-Actin was used as a loading control. Representative blots are shown. (B) Circular RNA levels were quantified using ImageQuant from three independent experiments and were normalized to the “β-gal, No copper” samples. Data are shown as mean ± SD. ** p<0.01, * p<0.05. (C) Northern blots were used to examine expression of HygroR transcripts when no CuSO₄ was added. (D) Summary of factors required for transcription termination of the HygroR mRNA [Model based on 52]. Depletion of shaded factors caused downstream circular RNA levels to increase, whereas depletion of unshaded factors had no or minimal effect.

Fig. 3.. Identification of factors required for transcription termination in human cells.

(A) Schematic of the pCRII-TOPO CMV-cGFP-SV40 pA + Laccase2 Exon 2 expression plasmid. Readthrough of the SV40 poly(A) signal can result in the production of the laccase2 circular RNA. (B) HeLa cells were transfected with the indicated siRNAs followed by plasmid transfection and total RNA isolation (48 h after siRNA transfection, 24 h after plasmid transfection). Northern blots were used to examine expression of the laccase2 circular RNA and the mature cGFP mRNA. *Concatenated and/or intertwined circular RNA. #Cross-reactive band that was observed when no plasmid was transfected. (C) Laccase2 circular RNA levels were quantified using ImageQuant from four independent Northern blot experiments and were normalized to the Mock samples. Data are shown as mean ± SD. * p<0.05.