Fig. 3. taRNAs constituted of truncated effector domains increase target protein level and manipulate cellular function.
a Schematic of HCV IRES domains. Superscript numbers on nucleotides indicate their position on the HCV genome. U228 (red) is critical for eIF3 binding. b Evaluation of effector domains via dual-luciferase assay. The conserved apical domains from HCV, CSFV, and PTV-1 IRES (blue) were attached to Fluc-targeting gRNA (g3′-1). HCV-IIIabcU228C (light gray) has an inactivating U228C mutation in the HCV-IRES IIIabc domain. Data were normalized to empty vector. n = 4 biological replicates. c Schematic of PTV-1 IIIab-based taRNA, with annotated domains and nucleotide sequence. d GAG-to-UGU mutations were introduced at the middle of Fluc-targeting gRNA in the PTV-IIIab taRNA (mis-g3′-1). This mismatched taRNA (light gray) was evaluated by dual-luciferase assay, along with original positive control (g3′-1, blue) and negative control (NT, dark gray). n = 4 biological replicates. e Representative western blot showing increased PTEN expression in HEK293T cells following transfection with g2(PTEN)-PTV-IIIab taRNA. GAPDH is the loading control. n = 7 biological replicates. f Representative western blot showing PTV-IIIab taRNA-mediated increase of SYNGAP1 protein in N2a cells. Two mSYNGAP1-targeting gRNAs (g1 and g2) were tested. α-tubulin is the loading control. n = 4 biological replicates. g Representative western blot showing PTV-IIIab taRNA-mediated increase of PMP22 protein in NIH/3T3 cells. Two mPMP22-targeting gRNAs (g1 and g2) were tested. GAPDH is the loading control. n = 3 biological replicates. h Dual upregulation of anti-proliferative proteins PTEN and p21 (gene: CDKN1A) by PTV-IIIab taRNAs inhibits MDA-MB-231 cell growth by 54%, measured by CCK-8 assay at 72 h after plating. n = 6 biological replicates. All bar-graph values are shown as mean ± SEM with data points. Statistical analyses were performed using one-way ANOVA with Sidak’s multiple comparisons test b vs. vector, and HCV-IIIabc vs HCV-IIIabcU228C; d vs. vector, mis-g3′-1 vs. NT, and mis-g′-1 vs. g3′-1; f–h Dunnett’s multiple comparison test vs. NT. Unpaired two-tailed Student’s t test was performed in (e). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The P value and source data are provided as a Source Data file.