IRES–cargo interplay structurally modulates circular RNA translation

Recent studies on endogenous circular RNAs (circRNAs) have revealed their high stability and unique conformation. 1 , 2 , 3 Engineered in vitro circularized RNAs (ivcRNAs), which serve as advanced platforms for sustained protein expression, 4 , 5 exhibit greater stability and lower immunogenicity than their linear counterparts. 4 , 5 , 6 , 7 , 8 Because ivcRNAs lack a 5′ m⁷G cap structure, they typically rely on a structured internal ribosome entry site (IRES) to initiate cap-independent translation, a process that depends on the distinct secondary and tertiary structures of viral IRESs. However, whether — and to what extent — cargo sequences within the ivcRNA platform affect IRES structure and translational efficiency remains poorly understood. Here, we demonstrate that extensive interactions between the Salivirus A IRES ( SV-A ) and certain cargo sequences compromise IRES structural integrity, thereby impairing translation. Correspondingly, disrupting unfavorable SV-A –cargo pairing restores efficient ivcRNA translation. To evaluate functional IRES elements for circular RNA translation, we engineered viral IRESs together with a firefly luciferase ( fLuc ) cargo sequence into the Anabaena transfer RNA (tRNA) Leu -derived 27-nt permuted intron–exon (PIE) system (Ana_PIE_27 nt; Fig. 1a ). 7 In total, 45 IRES elements derived from the 5′ untranslated regions (UTRs) of various viruses were examined, 9 , 10 and were theoretically categorized into five types based on sequence length, homology, and secondary structure (Fig. 1b ; Supplementary information, Table S1 ). Prior to transfection into various cell types for translation analysis, each ivcRNA was generated by in vitro transcription and self-catalytic circularization, and subsequently validated by denaturing urea–PAGE (polyacrylamide gel electrophoresis) (Supplementary information, Fig. S1 ). Using the fLuc–nLuc reporter assay (Fig. 1a ), type V IRESs generally exhibited superior translation efficiency (Fig. 1c ). Specifically, translation driven by type V IRESs was significantly higher than that of types I/II/III/IV in HeLa and C2C12 cells, and higher than that of types III/IV in 293FT and Huh-7 cells (Fig. 1c ). Notably, the type V SV-A displayed overall higher translation efficiency than the commonly used type I Coxsackievirus B3 IRES ( CVB3 ), 4 , 11 , 12 although translation efficiency varied across the tested cell lines (Fig. 1c ). Fig. 1: IRES–cargo interactions in ivcRNAs hinder the translational efficiency of SV-A -driven cargo sequences. a – c Screening of functional IRESs for ivcRNA translation. Using the fLuc reporter assay ( a ), different types of IRESs ( b ) were screened for their ability to drive ivcRNA translation ( c ). Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc test. ns not significant ( P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). d Schematic illustrating key factors for sequence optimization in the ivcRNA translation platform. TSS, translation start site. Y, yes; red Y indicates a newly introduced parameter for ivcRNA design. e Western blot analysis of codon-optimized gE expression from SV-A -driven ivcRNAs. f – h CircSHAPE-guided secondary structure models of SV-A in ivcRNAs carrying mCherry ( f ), gE_pre ( g ), or gE_jcat ( h ). i – n Restoration of SV-A domain IV structure and gE expression by disrupting IRES–cargo base pairing. Secondary structures of SV-A in various engineered ivcRNAs ( i ) were modeled for ivc-SV-A-gE_jcat mut 216 ( j ) and ivc-SV-A mut4-gE_jcat ( k ). Computational analysis of the IRES Crosstalk Ratio ( l ) revealed changes in SV-A –cargo interactions ( m ). Western blot analysis detected gE protein expression from various engineered ivcRNAs in 293FT cells ( n ). mut, mutant. See Supplementary information, Fig. S6 , for SV-A structures in additional ivcRNAs. In e , n , NC indicates the negative control (transfected with EGFP mRNA only). In i , dotted lines denote IRES–cargo interactions, and red boxes highlight mutated regions within the IRES. In f , g , h , j , k , domain IV is indicated in magenta. Full size image