Fig 1: CircAnks1a binding to Vegfb facilitated the recruitment of YBX1 to the Vegfb promoter. a The simulation showed a potential YBX1 binding site in the Vegfb gene promoter region at position −1843/−1831. b Chromatin immunoprecipitation assays were performed using YBX1 antibody on day 14 after SNL with or without circAnks1a siRNA application (i.t.) (**P < 0.01 vs. sham group, #P < 0.05 vs. SNL + scramble siRNA group, two-tailed one-way ANOVA, n = 4). c YBX1 overexpression enhanced luciferase activities, while circAnks1a overexpression had no effect on luciferase activity. (**P < 0.01 vs. control vector, #P < 0.05 vs. circRNA control, two-tailed one-way ANOVA, n = 6). d CircAnks1a expression in the cytoplasm and nucleus was examined on days 7 and 14 after SNL (**P < 0.01 vs. cytoplasm in sham group, #P < 0.05, ##P < 0.01 vs. nucleus in sham group, two-tailed one-way ANOVA, n = 5). e Dual-luciferase reporter assays showed that circAnks1a promoted the transcription of Vegfb by YBX1 via position −1834 to −1687 (promoter region 1). T1 truncation of promoter region 1, T2 truncation of promoter region 2 (**P < 0.01 vs. circRNA control group, ##P < 0.01 vs. the corresponding Vegfb-luc group, two-tailed one-way ANOVA, n = 6). f CircAnks1a markedly bound to the Vegfb promoter at position −1834 to −1687 (promoter region 1) in circAnks1a-overexpressing C6 cells (**P < 0.01 vs. mAnks1a probe group, two-tailed two-sample t tests, n = 3). g Binding of circAnks1a to the Vegfb promoter at position −1834 to −1687 was significantly increased following SNL (**P < 0.01 vs. corresponding mAnks1a probe group, #P < 0.05 vs. sham group, two-tailed two-sample t tests, n = 3). h Simulated diagram showing the binding sites between circAnks1a and Vegfb (red) and the binding sites between YBX1 and Vegfb (green). The data are presented as the mean ± s.e.m. Source data are available as a Source Data file
Fig 2: Increased YBX1 in the nucleus contributes to VEGFB expression. a Representative image labeled for YBX1, VEGFB, and DAPI. Scale bar, 50 μm (n = 3). b Continuous intrathecal administration of YBX1 siRNA prevented the upregulation of VEGFB mRNA induced by SNL (*P < 0.05, **P < 0.01 vs. sham group, ##P < 0.01 vs. corresponding scramble group, two-tailed one-way ANOVA, n = 5). c YBX1 siRNA (i.t.) attenuated the VEGFB protein upregulation on day 14 after SNL (*P < 0.05, **P < 0.01 vs. sham group, #P < 0.05 vs. corresponding scramble group, two-tailed one-way ANOVA, n = 4). d Suppression of transportin-1 by transportin-1 siRNA (i.t.) decreased YBX1 accumulation in the nucleus (**P < 0.01 vs. sham group, ##P < 0.01 vs. corresponding scramble group, two-tailed one-way ANOVA, n = 4). e, f Intrathecal injection of transportin-1 attenuated VEGFB upregulation on day 14 after SNL (*P < 0.05, **P < 0.01 vs. sham group, #P < 0.05 vs. corresponding scramble group, two-tailed one-way ANOVA, n = 5 for (e) and n = 4 for (f)). The data are presented as the mean ± s.e.m. Source data are available as a Source Data file
Fig 3: Model of the AR and NSUN2 positive feedback loop. AR pre‐mRNA is modified by NSUN2 and recognized by YBX1 and maintains its stability. At the same time, AR can act as a transcription factor to regulate the expression of NSUN2. In this way, a positive feedback loop is formed between NSUN2 and AR to promote the progression of prostate cancer. AR inhibitors can inhibit the transcriptional capacity of AR, but not AR alternative spliceosomes, such as AR‐V7. NSUN2 can regulate the stability of AR, and inhibition of NSUN2 in combination with AR inhibitors may better inhibit the progression of prostate cancer
Fig 4: AKT SILAC kinase screen. (a) In-vitro kinase reaction steps between either no kinase (control, CTL), recombinant active kinase (active kinase, AK) or heat-treated kinase (denatured kinase, DK) with recombinant protein substrates. Up to four reaction conditions were tested (30 min and 60 min incubations, each in the presence of 0.5 mM ATP or 1 mM ATP). The resulting reaction mixtures were separated by SDS-PAGE and analyzed by western blot or further processed with in-gel digestion followed by liquid chromatography tandem mass spectrometry (LC–MS/MS). (b) The reaction between AKT1/YB1 was validated by immunoblot of known phospho-sites on active AKT1 (T-308), and the AKT1-mediated YB1 phospho-site (S-102). Pan-AKT was used to show the absence, presence and denaturing effect on active AKT-1, while Total YB1 was used to show the presence of YB1 substrate in each reaction. (c) Phospho-peptides identified by LC–MS/MS were analyzed by label-free quantitation. The abundance of phospho-peptides resulting from the AKT1/YB1 reaction (0.5 mM ATP, 60 min) are represented as a heatmap (mean of two independent reactions). Square brackets indicate peptide locations within YB1 on which each phosphorylation was found. Hashtags denote a co-modification within that peptide, e.g. carbamidomethylation, deamidation, and/or oxidation. (d) Graphical summary explaining the SILAC based whole cell lysate (in vitro) kinase assay. (e) Volcano plot of all identified phospho-peptides identified from the AKT2 SILAC kinase screen. A minimal Log2 Fold change cut-off < 0.5 (red shading) was taken as potential increased phosphorylation events. (f) STRING cluster analysis49 of proteins corresponding to increased phospho-peptides identified in E. (g) KEGG pathway analysis50 of proteins identified in F. (h) Phospho-peptides with a Log2 Fold change > 1.0 were aligned using WebLogo v3.7.452. Residue colors indicate chemical properties of amino acids.
Fig 5: YBX1 is the reader of AR mRNA m5C sites. (A) Scatterplot of YBX1 expression and AR expression in the TCGA cohort. (B) Scatterplot of ALYREF expression and AR expression in the TCGA cohort. (C) qPCR analysis of the mRNA stability of AR, shNSUN2‐2 was used in the shNSUN2 group. (D) Nuclear and cytoplasmic mRNA were extracted, and AR mRNA was detected by qRT‐PCR in C4‐2 cells with or without NSUN2 knockdown or OE. (E) K‐M plot of the disease‐free survival of PCa patients with high or low YBX1 expression in the TCGA cohort. (F) Assessment of the binding abilities of YBX1 with AR mRNA in C4‐2 cells by YBX1‐RIP qPCR (n = 3 independent experiments). The p values were determined using a two‐sided unpaired student's t‐test. (G) Assessment of the HDGF m5C modification sites in C4‐2 cells by YBX1‐RIP qPCR (n = 3 independent experiments). The p values were determined using a two‐sided unpaired student's t‐test. (H) Silencing YBX1 in C4‐2 cells decreased AR mRNA expression (n = 3 independent experiments). The p values were determined using a two‐sided unpaired student's t‐test. (I) Assessment of the ability of YBX1 protein (200 ng) to bind with modified (the right line) or unmodified (the left line) AR site 2 probes (20 ng) by EMSA (n = 3 independent experiments)
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