Fig. 7: KPNA2/YBX1 axis regulated DDX3X intron retention and RNA stability.
A Sashimi plots of two osteosarcoma cell lines showed that DDX3X intron retention (IR) decreased after YBX1 knockdown. The DDX3X IR introduced premature termination codons (PTCs) that induce nonsense-mediated mRNA decay. The IR ratios and YBX1 binding motif were indicated. B The IgG and YBX1 groups were subjected to RIP followed by qPCR (RIP-qPCR) analysis to target DDX3X intron 17 (n = 3). C The interaction of YBX1 protein and DDX3X promoter was identified using the ChIP-qPCR assay and assessed by relative enrichment to input (n = 3). D qRT-PCR for DDX3X spliced and IR transcripts, following nuclear fractionation of U2OS cell lysates after YBX1 knockdown (n = 3). E qRT-PCR for DDX3X IR/mRNA transcript level with or without NMD inhibitor (NMDI14) treatment regulated by KPNA2/YBX1 axis (n = 3). F The half-live time (t1/2) of DDX3X mRNA was assessed after Actinomycin D (ActD) treatment regulated by KPNA2/YBX1 axis (n = 3). G Western blotting showed the DDX3X protein levels in U2OS and MNNG/HOS cell lines regulated by the KPNA2/YBX1 axis (left panel), with quantification of DDX3X expression normalized to GAPDH shown in the right panel (n = 3). ns no statistical significance; *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Two-tailed paired t tests for (A). One-way analysis of variance (ANOVA) was used to compare the difference in (B–E, G). Data were presented as mean ± SD (error bars) and representative of three independent experiments in (A–G).
