Fig 2: DDX5 gene is co-amplified with MIR21 and DDX5 facilitates maturation of pri-miR-21. a Immunoprecipitation (IP) and western blotting analyses were performed using indicated antibodies. Normal immunoglobulin G (IgG) was used as a negative control for IP. The RNA-binding protein DDX1 was used as a positive control for the Drosha-binding proteins. b The DDX5-bound pri-miRNAs were immunoprecipitated with DDX5 and analyzed by qRT-PCR. Control IgG was used as a negative control. c Levels of primary or mature forms of miR-21 were analyzed in control and DDX5-knockdown breast cancer cells harboring 17q23 amplicon. d Levels of mature miR-21 and DDX5 were analyzed in breast tumor samples using in situ hybridization and immunohistochemistry. Representative staining images of tissue samples are shown. Scale bar, 100 μm. e Soft agar colony formation assays with MMTV-ErbB2 mouse mammary epithelial cells transduced with control vector or lentiviral vector expressing the indicated genes. Representative images are shown in the middle panel and relative expression levels of mature miR-16 or miR-21 are shown in the right panel. *p < 0.05; **p < 0.01; ***p < 0.001; unpaired 2-tailed t-test (b, c and e). Data are presented as mean ± SD and are representative of 3 independent experiments

Fig 3: DDX3X interacts with Drosha/DGCR8 complex and affects the pri-miRNA and mature miRNA expression levels.(A) The interactions between DDX3X and Drosha complex tested by immunoprecipitation (IP) experiment. The uncropped full-length blots were presented in Figure S3. (B) The interactions between DDX3X and Dicer tested by immunoprecipitation (IP) experiment. The uncropped full-length blots were presented in Figure S3. (C) Relative pri-miRNA expression levels of DDX3X-dependent miRNAs (miR-1, miR-141, miR-145, miR-19b, miR-20a and miR-34a) in DDX3X KD cell lines compared with negative control (NC) cell lines. The DDX3X-independent miRNA (miR-21) was used as an internal control. The asterisk(s) (*,**) indicate a significant difference (p < 0.05, p < 0.01) respectively. (D) Relative mature miRNA expression levels of DDX3X-dependent miRNAs in DDX3X KD cell lines compared with NC cell lines. The DDX3X-independent miRNA (miR-21) was used as an internal control. The asterisks (**) indicate a significant difference (p < 0.01). (E) Relative pri-miRNA expression levels of DDX3X-dependent miRNAs in DDX3X OE cell lines compared with NC cell lines. The DDX3X-independent miRNA (miR-21) was used as an internal control. The asterisk(s) (*,**) indicate a significant difference (p < 0.05, p < 0.01) respectively. (F) Relative mature miRNA expression levels of DDX3X-dependent miRNAs in DDX3X OE cell lines compared with NC cell lines. The DDX3X-independent miRNA (miR-21) was used as an internal control. The asterisk(s) (*,**) indicate a significant difference (p < 0.05, p < 0.01) respectively.

Fig 4: Dicer interacts with p53 both in vitro and in vivo.(A) Schematic diagram of the p53 domain. TAD, transactivation domain; PRD, proline domain; TD, tetramerization domain; CTD, C-terminal domain. (B) Count of peptide of p53 interacted protein was shown as a table. (C) Western blot analysis of the interaction between overexpressed p53 and Dicer in H1299 cells. IP, immunoprecipitation. (D) In vitro binding assay of GST-p53 amino acids 1 to 100, amino acids 101 to 300, or amino acids 301 to 393 and purified Dicer. (E and F) Western blot analysis of p53 and Dicer domains for their interaction in H1299 cells. FL, full length, amino acids 1 to 1922; NT, N-terminal, amino acids 1 to 626; M, middle, amino acids 627 to 1270; CT, C-terminal, amino acids 1271 to 1922; ∆, full-length Dicer lacking, amino acids 1379 to 1551. (G to I) Western blot analysis of endogenous interaction among p53, Dicer, Drosha, and DGCR8 in HCT116 (G), U2OS (H), or A375 (I) cells. (J and K) Western blot analysis of endogenous interaction between Dicer and p53 in HCT116 (J) or U2OS (K) cells. (L) Western blot analysis of the interaction between p53 and Dicer in the cytoplasm (Cyto.) or nucleus (Nucl.) fraction of HCT116 cells upon MG132 treatment. All data are shown as representative of three experiments.

Fig 5: DDX3X facilitates pri-miRNAs binding to Drosha/DGCR8 complex and elevates the processing activity of Drosha/DGCR8 complex on pri-miRNAs.(A) The binding affinity of DDX3X-dependent pri-miRNAs with DDX3X tested by RNA immunoprecipitation (RIP) experiment. The DDX3X-independent pri-miRNA (pri-miR-21) was used as an internal control. The asterisk(s) (**,***) indicate a significant difference (p < 0.01, p < 0.001) respectively. (B) The binding affinity of DDX3X-dependent pri-miRNAs with DDX3X in DDX3X KD cell lines tested by RNA immunoprecipitation (RIP) experiment. The DDX3X-independent pri-miRNA (pri-miR-21) was used as an internal control. (C) The binding affinity of DDX3X-dependent pri-miRNAs with Drosha in DDX3X KD#1, KD#2 and their NC cell lines tested by RNA immunoprecipitation (RIP) experiment. The DDX3X-independent pri-miRNA (pri-miR-21) was used as an internal control. The asterisks (**) indicate a significant difference (p < 0.01). (D) A schematic model for in vivo monitoring of pri-miRNA processing. (E) Relative luciferase activities of DDX3X-dependent pri-miRNA pmirGLO vectors in DDX3X KD#1, KD#2 and their NC cell lines tested by dual luciferase reporter assay. The DDX3X-independent pri-miRNA (pri-miR-21) and empty vector pmirGLO were used as internal controls. The asterisks (**) indicate a significant difference (p < 0.01). (F) Relative luciferase activities of DDX3X-dependent pri-miRNA pmirGLO vectors in DDX3X OE and its NC cell lines tested by dual luciferase reporter assay. The DDX3X-independent pri-miRNA (pri-miR-21) and empty vector pmirGLO were used as internal controls. The asterisk(s) (*,**) indicate a significant difference (p < 0.05, p < 0.01) respectively.