Fig. 7: Synergistic effects of PSPC1, RNA and NONO-TFE3 on transcription. | Nature Communications

Fig. 7: Synergistic effects of PSPC1, RNA and NONO-TFE3 on transcription.

From: RNA-mediated condensation of TFE3 oncofusions facilitates transcriptional hub formation to promote translocation renal cell carcinoma

Fig. 7

a In vitro kinase assay using recombinant proteins as indicated. Phosphorated CTD (p-CTD) and other components were determined by immunoblotting using their corresponding antibodies. n = 3 independent biological replicates. b Schematic illustration of the in vitro transcription assay. Created in BioRender. Suris, A. (https://BioRender.com/i1s8xt7). c Images of droplets formed during the in vitro transcription reaction. Hoechst was used to stain the DNA component within the droplets. Scale bar, 10 µm. n = 3 independent biological replicates. d Quantifications of in vitro transcription reaction activity (normalized to RNAPII only) with the indicated recombinant proteins. (n = 6 independent biological replicates; two-sided unpaired Student’s t-test). Data are shown as mean ± SD. e Schematic illustration of the luciferase reporter assay for assessing gene transcription. Constructs encoding the truncated NONO-TFE3 variants fused with the Gal4 DNA binding domain (Gal4DBD) were co-transfected into HEK293T cells bearing a luciferase reporter and containing 5 repeated GAL4-responsive upstream activating sequences (UAS) in the presence or absence of PSPC1. f Quantification of luciferase signals (normalized to the control) using NONO-TFE3 truncated variants or the TFE3 S321A mutant, with or without PSPC1. (n = 4 independent biological replicates; two-sided unpaired Student’s t-test). Data are shown as mean ± SD. g Schematic diagram showing the interplay among NONO-TFE3, PSPC1 and RNA in promoting droplet formation and subsequent CTD incorporation and phosphorylation. RNAPII CTD alone exhibits limited ability to form condensates. Co-condensation with NONO-TFE3 and PSPC1 enhances droplet formation, resulting in larger condensates. RNA further cooperates with NONO-TFE3 and PSPC1 to markedly promote condensate assembly and CTD incorporation, thereby concentrating CTD and CDKs to facilitate more efficient phosphorylation. Source data are provided as a Source Data file.

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