Fig. 6: Identification of Gua-responsive riboswitches based on a U1-snRNP-dependent mode of action.

a Secondary structure of the Gua-U1-snRNP riboswitch library design. The U1-snRNP binding site (CAGGUAAGU) is included into a prolonged stem P1 of the guanine aptamer. Blue nucleotides indicate the randomized motif. b Mode of action of U1-snRNP-dependent riboswitches. In the ligand-unbound conformation, the U1-snRNP binding site is available, allowing for U1-snRNP binding and interference with the polyadenylation process. Upon ligand binding, the aptamer stem is stabilized and the U1-snRNP binding site is masked, enabling polyadenylation and subsequent gene expression. c Guanine riboswitch construct frequency analysis of the original library. d Volcano plots showing the significance (FDR) of changes in riboswitch abundance as a function of the change in expression (log₂ fold change). e Selected hits from the Gua-U1-snRNP riboswitch library screen, randomized sequence, mean counts per million (CPM), fold change (FC) and false-discovery rate (FDR) at 30, 100, and 300 µM guanine, respectively. The bar plots show the raw counts under untreated, 30, 100, and 300 µM µM Gua-stimulated conditions (four groups from left to right, n = 8 replicates each). f Functional hit validation in HEK-293 cells at increasing guanine doses [µM] (n = 3 replicates, mean ± s.d.); U1-bs: construct harboring the freely accessible U1-snRNP binding site. g Maximal fold changes and p-values measured upon stimulation vs. 0 µM guanine for all tested constructs. *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired T-Test, two-tailed). Source data are provided as a Source Data file.