Fig. 7

FUS promotes the biogenesis of circEHD2. A, Schematic illustration of circEHD2 pre-mRNA pull-down following mass spectrometry (MS) to identify circEHD2 splicing factors. B, MS analysis of splicing factors that interacted with circEHD2 pre-mRNA via RNA pull-down assay. C and D, qRT–PCR analysis of the level of FUS and circEHD2 in RCC cells when transfected with si-FUS. E and F, qRT–PCR analysis the level of FUS and circEHD2 in RCC cells when overexpression of FUS. G, The putative binding sites of FUS in the upstream and downstream of circEHD2 pre-mRNA were predicted by catRAPID. H, The RIP assay was performed to confirm the binding sites of FUS on circEHD2 pre-mRNA in OSRC-2 cells. miR-200 was used as the positive control. I, The RNA pull-down assay analyzed the interaction between FUS and above 10 fragments of circEHD2 pre-mRNA (P1-P10) in OSRC-2 cells. J, Dual-luciferase reporter assays showed that FUS could bind to circEHD2 pre-mRNA (-432-nt to -143-nt). K, Representative images of IHC and ISH showed the expression of FUS and circEHD2 in ccRCC tissues and paired NATs (n = 80). Scale bars: 50 μm. L, The H-score of FUS in ccRCC tissues and paired NATs (n = 80). M, The Pearson correlation analysis showed that the level of FUS was positively correlated with that of circEHD2. N, The level of FUS was upregulated in kidney renal clear cell carcinoma from the TCGA database. Error bars represent the standard deviation (SD) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001