Fig 2: 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 3: 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.

Fig 4: USP36 does not affect the levels of DGCR8 and Drosha. A, Overexpression of USP36 does not increase the levels of DGCR8 and Drosha. H1299, 293, or U2OS cells were transfected with WT USP36 or its catalytically-inactive C131A (CA) mutant and assayed for protein expression by IB. Knockdown of USP36 does not reduce the levels of DGCR8 and Drosha. H1299 and 293 cells transfected with scr or USP36 siRNA (B) and H1299 cells infected with scr or USP36 shRNA (C) were assayed by IB.

Fig 5: S677 DGCR8 phosphorylation is critical for the recruitment of RNF168 to RNF8 and MDC1, DGCR8-USP51 interaction, DGCR8 deubiquitination, and histone ubiquitination upon irradiation.a, b MYC-GFP-, WT DGCR8-, S677A-DGCR8-, and S677D-DGCR8-overexpressing LM2 cells with or without IR treatment (a, 8 Gy followed by 1-h incubation; b, 8 Gy followed by 8-h incubation) were subjected to pulldown with MYC beads and immunoblotting with the indicated antibodies. c HEK293T cells with stable overexpression of MYC-tagged WT DGCR8, S677A-DGCR8, or S677D-DGCR8 were co-transfected with SFB-USP51 (WT or the C372S mutant) and HA-tagged ubiquitin, and then treated with IR (8 Gy). After 8 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. d Quantification of ?H2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8, S677A-DGCR8, or S677D-DGCR8. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against ?H2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. 9). n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t-test. Error bars are mean ± SEM. e DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8 or the S677A mutant were transduced with FLAG-H2A and RNF8 or RNF168. The cells were then transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. LE long exposure, SE short exposure. Source data are provided as a Source Data file.