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. 2021 Aug 20;12:5057. doi: 10.1038/s41467-021-25078-5

Fig. 7. Disrupted RBSs or RBP knockout reduce the target-site accessibility to AGO.

Fig. 7

a 3′UTR structures of RNA substrates (SSB, UBA1, and CRAT) used for gel mobility-shift assays shown in b–d. The 3′UTRs of SSB and UBA1 were also used as targets for luciferase assays in Fig. 8a, b. The miRNA target sites (MTSs) and RBP-binding sites (RBSs) are shown as red and blue boxes, respectively, with the mutated positions indicated by orange boxes. b Histidine-tagged recombinant RBPs (His-FUBP3 or His-PCBP2) and GST proteins were used for gel mobility-shift assays shown in (c, d). c Gel mobility-shift assays for RBSWT and RBSMUT with His-FUBP3 or His-PCBP2 with GST protein used as a negative control (top). The free RNA and RNA:RBP complex bands are shown as black and blue rectangles, respectively. Mean fractions of the bound RNA:RBP complexes ±95% confidence intervals are displayed (bottom, n = 3). d Gel mobility-shift assays with (1) 3′UTR, (2) 3′UTR and rhAGO2, (3) 3′UTR and RBP, and (4) 3′UTR, RBP, and rhAGO2. Otherwise as in (c). e DMS reactivities on A and C nucleotides of 3′UTRs were measured by comparing DMS counts of DMS(−) and DMS(+) samples. Corresponding nucleotides were divided into three groups by the RBP-binding signal of IGF2BP1 eCLIP-seq dataset (No RBS, Lenient, and Stringent). DMS reactivities between WT and RBP KO were compared (two-sided Wilcoxon’s rank-sum test). The mean DMS reactivity values ±95% confidence intervals are displayed. f Experimental procedure for AGO2-immunoprecipitation (IP) followed by western blot and RT-qPCR. g AGO2-IP followed by western blot and RT-qPCR in AGO2-overexpressed HEK293T parental and RBP KO (IGF2BP1 or PCBP2) cells. Protein levels in the input and IPed samples were visualized by the western blotting (left). Relative RNA levels of each input and IPed samples were quantitated in parental and RBP KO cells and normalized to each input sample (right, two-sided Wilcoxon’s rank-sum test, *P < 0.05, **P < 0.01, ***P < 0.001, n = 9 for PCBP2 and n = 6 for IGF2BP1). The mean relative RNA level of the IPed samples ±95% confidence intervals are displayed. The RNA level of KATNA1 was used as a negative control and U6 snRNA-level served as a technical control of AGO2-IP. P values are provided in Source Data.