FUS interacts with TARDBP RNA and regulates translation through the MP20 3′UTR. (A) Schematic representation of UV cross-linking (UV Xlink) and IP followed by RT-PCR. UV Xlink was performed, and IP using an anti-FUS antibody isolated the FUS–RNA complex from HEK293T cells. Subsequent RT-PCR analysis showed whether endogenous FUS associates with TARDBP RNA. (B) Schematic diagram of FUS-binding consensus sequences on TARDBP exon 6. Green circles represent GGUG (Iko et al., 2004; Lerga et al., 2001), and dark green circles represent GUGGU (Lagier-Tourenne et al., 2012). Detection of TARDBP RNA after UV Xlink and IP was achieved using primers targeting exon 6 (410–566, black bold line). Orange and blue lines show alternative donor and acceptor sites, respectively, while magenta lines indicate the FL-stop. (C) UV Xlink followed by IP, and RT-PCR images showing IP validation. An anti-IgG antibody was used as a negative control for IP. (D) Quantification of FL-endo or SVs-endo protein levels from the WB analysis in Fig. 9 F. Data are normalized to β-actin. Statistical analyses were performed using one-way ANOVA followed by Tukey’s test (n = 3 for each group). (E) Schematic diagram of the MP20 3′UTR construct. TARDBP exon 6 (1,205–1,940) was inserted downstream of Venus, while Venus alone was used as control construct. (F and G) WB analysis showing the dose-dependent effect of FLAG-WT FUS (F) or FLAG-P525L FUS mutant (G) on each Venus reporter construct. Quantification of Venus in F and G is normalized to β-actin. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s test. All graphs show the mean ± SEM. *P < 0.05. Source data are available for this figure: SourceData FS5.