Journal: bioRxiv

Article Title: 3’ to 5’ Translation of Circular RNAs?

doi: 10.64898/2025.12.08.692888

Figure Lengend Snippet: (A) Schematic of vector design for backward translation of enhanced green fluorescent protein (eGFP) in human cells. The pcDNA3.1 vector was used as the backbone for circRNA expression. The construct consisted of two reverse eGFP fragments (coordinates: 593→1 and 720→594), separated by a translation initiation site (TIS)-containing sequence. The TIS sequence was flanked by a backward start codon (←GUA) and a backward stop codon (AAU←). TIS elements used in this study included either upstream sequences derived from endogenous backward ORFs or the EMCV internal ribosome entry site (IRES). (B) Structural illustration of two circ-(bw)-eGFP constructs: one lacking a TIS sequence element and one incorporating EMCV-IRES as TIS. The eGFP sequence is shown in green, and the TIS region is marked in red. (C) RT-qPCR quantification of reverse eGFP expression in HEK-293T cells transfected with empty vector, reverse eGFP without TIS, or reverse eGFP containing EMCV-IRES as TIS ( n =3). Tubulin was used as the internal reference gene. Primers spanning the back-splice junction (BSJ) were used to specifically detect circular RNA. All RNA samples were treated with RNase R to enrich for circular species. (D) Representative confocal microscopy images showing eGFP fluorescence (green) resulting from backward translation of circ-(bw)-eGFP in HEK-293T cells. Fluorescence was observed in cells transfected with reverse eGFP containing EMCV-IRES, but not in those lacking TIS. Scale bar, 20 μm. The fluorescent signal was detected in ∼1–2 out of every 100,000 cells. Live cells were stained with Mito-Tracker (red, mitochondria). (E) Summary of four sequence features known to promote circRNA translation upstream of backward ORFs: IRES-like elements predicted by IRESfinder, IRES-like hexamers aligned to the 10-nt region upstream of the backward start codon, Kozak consensus sequences identified by in-house scripts, and N6-methyladenosine (m⁶A) sites predicted using SRAMP. (F) The secondary structure of conserved motif ‘TCCCA…TGGGA’ identified from backward-translated circRNAs using MEME analysis. (G) Predicted secondary structures of circRNAs involved in regulating backward translation, modeled using RNAfold. From left to right: hs. circHSH2D; circ-(bw)-eGFP incorporating hs. circHSH2D-derived TIS; circ-(bw)-eGFP with the same TIS but with a stem-loop structure disrupted by mutation; and circ-(bw)-eGFP in which the backward start codon (5’-GUA-3’) was mutated to 5’-GCA-3’. The latter two constructs were predicted to be untranslatable. (H) RT-qPCR quantification of reverse eGFP in HEK-293T cells transfected with empty vector, hs. circHSH2D-TIS-(bw)-eGFP, hs. circHSH2D-TIS-(bw)-eGFP with stem-loop disruption, and hs. circHSH2D-TIS-(bw)-eGFP with start codon mutation (n=3). Tubulin was used as the internal control. Primers spanned the BSJ, and RNase R treatment was applied to enrich for circRNAs. (I) Representative confocal microscopy images showing eGFP fluorescence (green) from backward translation of circ-(bw)-eGFP in HEK-293T cells transfected with constructs listed in panel h. Fluorescence was observed only in cells transfected with hs. circHSH2D-TIS-(bw)-eGFP. No signal was detected in other groups. Scale bar, 20 μm. live cells were stained with Mito-Tracker (red, mitochondria). Statistical comparisons of eGFP expression levels under different conditions were performed using Mann-Whitney U in GraphPad Prism (version 9.2.0). Statistical significance: ns : not significant, “*”: p <0.05, “**”: p <0.01, “***”: p <0.001, “****”: p <0.0001.

Article Snippet: The plasmids for human cell lines in this study were generated using pcDNA3.1(+) ZKSCAN1 MCS + Sense IRES (Addgene: #69909) and pcDNA3.1(+).

Techniques: Plasmid Preparation, Expressing, Construct, Sequencing, Derivative Assay, Quantitative RT-PCR, Transfection, Confocal Microscopy, Fluorescence, Staining, Mutagenesis, Disruption, Control, MANN-WHITNEY