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. 2023 Oct 26;14:6827. doi: 10.1038/s41467-023-42252-z

Fig. 1. taRNAs built from an array of IRESs increase reporter gene translation.

Fig. 1

a Schematic overview of taRNA technology. taRNAs recruit initiation factors (eIFs, blue) to increase targeted mRNA translation. A taRNA molecule is made of a target-specific guide RNA domain (gRNA, green), a linker (gray) and a translation machinery-recruitment domain (purple). Poly(A)-binding protein (PABP, light gray) interacts with both eIFs and mRNA poly-A tail. b Schematic of vectors for taRNA and reporter used in dual-luciferase assay. The taRNA (green and purple) targets Firefly luciferase mRNA (Fluc, blue), while Renilla luciferase (Rluc, gray) is the internal control. The nucleotides at position −4 to −1 before the start codon of Fluc mRNA are ATTG. The hU6 (human U6), PGK (phosphoglycerate kinase 1) and SV40 (simian vacuolating virus 40) indicate different promoters. c Evaluation of viral IRESs as taRNA effector domains in HEK293T cells by dual-luciferase assay. Each IRES was attached to either a non-targeting gRNA (gray), or an Fluc-targeting gRNA (g3′-1, blue) on the taRNA vector, and co-transfected into HEK293T cells with the reporter. Viral origin and classification are listed for each IRES. Data were normalized to empty vector group. n = 8 biological replicates for empty vector group. n = 4 biological replicates for all the other groups. d RNA level of Fluc relative to Rluc in HEK293T cells after taRNA treatment (gRNA: g3′ -1, blue) measured by RT-qPCR. Data were normalized to empty vector group. n = 3 biological replicates. e PTV-based taRNAs with gRNAs that bind to different regions on Fluc transcript, including 5′ UTR (g5′), CDS (gCDS-1) and 3′ UTR (g3′-1), were evaluated by dual-luciferase assay. n = 4 biological replicates. All data are shown as mean ± SEM with individual data points. c Statistical analyses were performed using two-way ANOVA with Sidak’s multiple comparisons test between non-targeting and Fluc-targeting within each IRES group. Statistical analyses were performed using one-way ANOVA with Dunnett’s multiple comparison test d vs. vector; e vs. NT. **P < 0.01, ****P < 0.0001. No asterisk = not significant. The P value and source data are provided as a Source Data file.