Trans-spliced mRNA products produced from circRNA expression vectors

Abstract

Circular (circ) RNA expression vectors are used as a method of identifying and characterizing RNA sequences that harbor internal ribosome entry site (IRES) activity. During the course of developing a vector series tailored for IRES discovery, we found evidence for the occurrence of trans-spliced mRNAs arising when sequences with promoter activity were embedded between the upstream CTD and downstream NTD exons of the pre-mRNA. These trans-spliced products regenerate the same open reading frame expected from a circRNA and can lead to false-positive signals in screens relying on circRNA expression vectors for IRES discovery. Our results caution against interpretations of IRES activity solely based on results obtained from circRNA expression vectors.

Keywords: IRES; circRNA; gene expression; trans-splicing.

FIGURE 1.

The PGK promoter, as test sequence in a circRNA expression vector context, leads to reporter expression. (A) Schematic diagram of circv1. The positions of the inverted Alu repeats with the two introns are denoted by violet arrowheads. (B) Relative RLuc expression obtained from 293T cells following transfection with circv1 harboring test elements from the indicated genes (Supplemental Table S1). Mock; mock transfection. Values are set relative to those obtained with EMCV Scrb J/K control sequences in circv1. n = 3 ± SEM. (C) Schematic illustrating potential mechanism by which a shortened RLuc_NTD-containing mRNA produced from the PGK promoter can participate in a trans-splicing reaction to generate a full-length, functional renilla ORF.

FIGURE 2.

CircRNA expression vectors can generate trans-spliced products. (A) Schematic representation of circv6GFP.PGK and circv6RLuc.PGK expression vectors. (B) Trans-spliced products expected from circv6RLuc.PGK and circv6GFP.PGK vectors. Only trans-spliced products formed from the CMV (full-length mRNA) and PGK (shorter mRNAs) promoters are shown due to space limitations, but we envisage homotypic (GFP and RLuc) products also being produced, as well as products from trans-splicing of two full-length, CMV-synthesized mRNAs. The relative location of PCR primers (RLuc-F; R_f, RLuc-R; R_r, GFP-F; G_f, and GFP-R; G_r) used for product detection are shown. (C) Endpoint RT-PCR analysis of RNA isolated from 293T cells transfected with the indicated expression plasmids. RNA was isolated 48 h following transfection. (Top panel) PCRs performed on RNA samples that had not been converted to cDNA (−RT). (Bottom panel) RT-PCR products obtained after 25 cycles. (D) RT-qPCR analysis of RNA from 293T cells transfected with the indicated plasmids. The combination of plasmids used in the transfection, as well as primer pairs used in the qPCR, are shown below the graph. The relative abundance of each PCR product was calculated using the ΔΔCt method with GFP or RLuc signal calculated relative to GAPDH. n = 6 ± SD. (E) Sequencing chromatogram of the PCR product obtained from RNA of cells cotransfected with circv6GFP.PGK and circv6RLuc.PGK. PCR primers crossed the GFP(NTD)/RLuc(CTD) exon junction.

FIGURE 3.

Protein expression from trans-spliced mRNA products. (A) Schematic representation of circR/G.PGK and circG/R.PGK expression vectors. (B) Schematic diagram illustrating how trans-splicing of the indicated cotransfected vectors can lead to the production of full-length RLuc and GFP proteins. Due to space constraints, not all possible trans-splicing reactions are shown. (C) Luciferase values obtained from 293T cells cotransfected with the indicated expression vectors. RLuc RLU values were normalized to total protein levels in the extracts. n = 4 ± SD. (D) Flow cytometry showing representative GFP expression obtained from 293T cells transfected with the indicated individual or combinations of plasmids.