Fig 2: RAN increases TDP43 nucleus import and facilitates TDP43 binding G3BP1 mRNA.A, after treatment with actinomycin D (10 μg/ml), G3BP1 mRNA level was quantified at indicated times in control, RAN-silenced, TDP43-silenced, and both RAN and TDP43-silenced cells. The half-life of G3BP1 mRNA was analyzed by plotting degradation curves. Data are presented as the mean ± 95%CI (n = 3). B, TDP43 protein levels with knockdown of RAN were detected by Western blotting. The blots are the representation of three independent experiments. Data are presented as the mean ± SD (n = 3). C, relative enrichment of G3BP1 mRNA immunoprecipitated by anti-TDP43 or anti-IgG antibody was indicated by RT-qPCR upon knockdown of RAN. Data are presented as the mean ± SD (n = 3). D, the nucleus and cytoplasm proteins in HONE-1 and SUNE-1 cells were isolated by nuclear/cytosol fractionation assays. TDP43 distribution in the nucleus or cytoplasm was identified by Western blotting with or without RAN silencing, GAPDH as the cytoplasm marker, and Lamin B as the nucleus marker. The blots are the representation of three independent experiments. Data are presented as the mean ± SD (n = 3). E, TDP43-GFP overexpression vectors were transfected in HONE-1 and SUNE-1 cells after RAN silencing. Recognizing TDP43-GFP protein distribution (green) in HONE-1 and SUNE-1 cells after 24 h of TDP43-GFP overexpression vectors transfected, range indicator was performed to show clearer distribution of TDP43-GFP in the nucleus or cytoplasm, cell nuclei were stained with DAPI (blue). The scale bar represents 10 μm. F, the average fluorescence intensity of TDP43-GFP in the nucleus (lower) and cytoplasm (upper) of each cell was calculated. Data are presented as the mean ± SD (n = 20). ∗p < 0.05 and ∗∗p < 0.01. The significant differences were assessed using one-way ANOVA (B, D, and F), two-way ANOVA (A), and t test (C). RT-qPCR, reverse transcription quantitative real-time PCR; DAPI, 4′, 6-diamino-2-phenylindole; CI, confidence interval.

Fig 3: RAN, TDP43, and G3BP1 mRNA form a complex in the nucleus.A, diagrams of full-length G3BP1 transcript consist of CDS and 3′ UTR, as well as its deletion fragments. B and C, the in vitro–transcribed full-length G3BP1 transcript and deletion fragments with the correct sizes were indicated (upper). RNA pull-down assay and Western blotting showed whether these biotin-labeled G3BP1 fragments could pull down RAN in cell lysates (lower). D, the motif discovery algorithm DREME was used to recognize the top consensus motif based on the RIP-seq data. E, construction of mutant fragments based on predicted motifs. F, the in vitro–transcribed D2 fragments and mutant fragments with the correct sizes are indicated (upper). RNA pull-down assay and Western blotting indicated whether these biotin-labeled fragments could pull down RAN in cell lysates (lower). G, the interaction between endogenous RAN and TDP43 was evaluated by Co-IP assays using anti-RAN antibody or normal rabbit IgG in HONE-1 and SUNE-1 cell lysates. H, the interaction between endogenous TDP43 and RAN was evaluated by Co-IP assays using anti-TDP43 antibody or normal rabbit IgG in HONE-1 and SUNE-1 cell lysates. I, RAN (red) or TDP43 (green) protein distribution in cells was recognized by IF, and cell nuclei were stained with DAPI (blue). The scale bar represents 10 μm. J, relative enrichment of G3BP1 mRNA immunoprecipitated by anti-TDP43 or anti-IgG antibody was indicated by RT-qPCR. Data are presented as the mean ± SD (n = 3). K, enrichment of TDP43 pulled down by biotin-labeled G3BP1 probes from in vitro transcription or control antisense probes was detected by Western blotting. L, RNA pull-down assay and Western blotting showed whether biotin-labeled full-length G3BP1 transcript or deletion fragments could pull down TDP43 in cell lysates. M, TDP43 protein distribution in cells was recognized by IF (green), G3BP1 mRNA distribution in cells was recognized by FISH (red), and cell nuclei were stained with DAPI (blue). The scale bar represents 10 μm. N, in vitro purified RAN-GST, TDP43, eEF2-His (positive control), and GST (negative control) proteins were used to perform a cell-free RNA pull-down experiment. Coomassie brilliant blue staining showed the purification of these proteins (left). Enrichment of proteins pulled down by biotin-labeled G3BP1 probes from in vitro transcription was detected by Western blotting (right). O, in vitro purified RAN-GST, TDP43, and GST (negative control) proteins were used to perform a cell-free co-IP experiment. Coomassie brilliant blue staining showed the purification of these proteins (left). Enrichment of proteins immunoprecipitated by anti-GST magnetic beads was detected by Western blotting (right). ∗p < 0.05 and ∗∗p < 0.01. The significant differences were assessed using t test (J). RT-qPCR, reverse transcription quantitative real-time PCR; ; NPC, nasopharyngeal carcinoma; Co-IP, coimmunoprecipitation; GST, glutathione-S-transferase; IF, immunofluorescence; DAPI, 4′, 6-diamino-2-phenylindole; CDS, coding sequence.