Fig 1: USP51 binds and stabilizes DGCR8.a LM2 cells were treated with 10 µM MG132, irradiated with 8 Gy X-ray, and collected 6 h later. Lysates were immunoblotted with antibodies against DGCR8 and ß-actin. b Parental and radioresistant LM2 cells were treated with 100 µg/ml cycloheximide (CHX). Cells were collected at different time points and immunoblotted with antibodies against DGCR8 and ß-actin. c HEK293T cells were co-transfected with MYC-DGCR8 and HA-ubiquitin (Ub) or its lysine-specific mutants (K48, K63, K48R, or K63R), followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. Cells were treated with IR (8 Gy), followed by treatment with 10 µM MG132 for 6 h. 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. d SFB-tagged DUBs were individually co-transfected with MYC-DGCR8 into HEK293T cells, followed by pulldown with S-protein beads and immunoblotting with antibodies against FLAG and MYC. e HEK293T cells were co-transfected with MYC-DGCR8, HA-ubiquitin, and a candidate DUB. After treatment with MG132 for 6 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. f Immunoblotting of DGCR8, USP36, USP51, and ß-actin in LM2 cells with overexpression or knockdown of USP36 or USP51. g HEK293T cells were co-transfected with MYC-DGCR8 and SFB-tagged USP36 or USP51. After 48 h, cells were lysed, immunoprecipitated with anti-MYC beads, and immunoblotted with antibodies against FLAG and MYC. h Co-IP of endogenous DGCR8 with endogenous USP36 and USP51. DGCR8 was immunoprecipitated from LM2 cells and immunoblotted with antibodies against USP36, USP51, and DGCR8. i USP51 binds DGCR8 in vitro. Left panel: SFB-GFP or SFB-USP51 was retained on S-protein beads and incubated with purified MBP-DGCR8. The bound proteins were eluted by boiling in Laemmli buffer and immunoblotted with antibodies against MBP and FLAG. Right panel: purified SFB-GFP, SFB-USP51, and MBP-DGCR8 proteins were analyzed by SDS-PAGE and Coomassie blue (CB) staining. j Upper panel: LM2-R cells stably infected with USP51 shRNA were treated with 100 µg/ml CHX for the indicated times. Lysates were subjected to immunoblotting with antibodies against DGCR8, USP51, and ß-actin. Lower panel: DGCR8 levels were quantitated and normalized to ß-actin. Source data are provided as a Source Data file.
Fig 2: NKAP is a reader of m6A on pri-miR-25. a The NKAP-binding sites on pri-miR-25 detected by CIMS and CITS miCLIP-seq. Orange tracks of NKAP-iCLIP-seq are unique tag coverage and blue tracks represent C>T transition and truncation, respectively. Filled purple circles denote iCLIP-called NKAP-binding site and the horizontal blue bars indicate the transcript model of pre-miR-25. b The DGCR8-binding sites on pri-miR-25 detected by DGCR8 HITS-CLIP. Filled purple circles denote iCLIP-called NKAP-binding site and the horizontal blue and red bars represent the transcript model of pre-miR-25 and the DGCR8-binding site, respectively. c In vivo binding of NKAP-FLAG to pri-miR-25 in PDAC cells exposed to CSC or DMSO detected by CLIP-qPCR. Values are the mean ± S.D. from three independent experiments. d The levels of NKAP-FLAG bound to pri-miR-25 were significantly elevated by overexpression of METTL3, but significantly decreased by knockdown of METTL3 in both PANC-1 (left panel) and BXPC-3 (right panel) cells. Values are the mean ± S.D. from three independent experiments. e Data of m6A residues and NKAP-binding sites identified by the analysis of cross-linking-induced mutation sites (CIMS) or cross-linking-induced truncation sites (CITS) in iCLIP-seq data. f Venn diagram showing overlap between m6A residues and NKAP-binding sites. g Significant enrichment of RGAC motif is in NKAP footprints (CIMS or CITS along with 5-nucleotide flanking sequences) analyzed by MEME SUIT with default parameters. h Shown is the intensity of NKAP binding centered at m6A residues. i Shown is the intensity of m6A CLIP signal centered at NKAP-binding sites. j Shown is the distribution of NKAP iCLIP tag density around the splicing factor binding sites. k Shown is the binding intensity of NKAP at splicing sites near or not near m6A residues. All statistic examinations in this figure are Student t test unless specific. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding control
Fig 3: Overexpression of DGCR8 does not lead to apoptosis at E14.5 (A–D) Hoechst staining on coronal cryosections through the dorsal telencephalon of Dgcr8 WT (A) and Dgcr8 conditional knockout (cKO) (B) mouse embryos at E12.5 or on coronal cryosections through the dorsal telencephalon of Control (C) and DGCR8 OE (D) mouse embryos at E14.5 after IUEp at E12.5. (A'–D') Immunostaining for activated CASPASE-3 (green) on coronal cryosections through the dorsal telencephalon of Dgcr8 WT (A') and Dgcr8 cKO (B') mouse embryos at E12.5 or on coronal cryosections through the dorsal telencephalon of Control (C') and DGCR8 OE (D') mouse embryos at E14.5 after IUEp at E12.5; electroporated cells (mCherry, red); dashed lines indicate limits of the cortical wall (from outside to inside: NL: neuronal layer, IZ: intermediate zone, SVZ: subventricular zone and VZ: ventricular zone); scale bars: 20 µm (A–B'); scale bar: 100 µm (C–D'). *p-value < 0.05; **p-value < 0.01.
Fig 4: Overexpression of DGCR8 decreases the generation of upper-layer neurons and selectively promotes BP expansion at E16.5 (A,A') Immunostaining for CUX1 (green) and mCherry+ electroporated cells (red) on coronal cryosections through the dorsal telencephalon of Control and DGCR8 OE mouse embryos at E16.5 after IUEp at E14.5. (B) Quantification of the proportion of CUX1+ mCherry+ (A,A') cells expressed in % over total mCherry+ cells; scale bar 50 µm (A) and 20 µm (A'). VZ: ventricular zone, SVZ: subventricular zone, IZ: intermediate zone, NL: neuronal layer, LII-III: cortical layer 2 and 3, LIV-VI: cortical layer 4-5. Error bars indicate the variation of four Control and four DGCR8 OE electroporated cortices (s.e.m.); unpaired Student's t-test. (C,E) Immunostaining for PAX6 (C, green) or TBR2 (E, green) and mCherry+ electroporated cells (C,E, red) and merged images on coronal cryosections through the dorsal telencephalon of Control and DGCR8 OE mouse embryos at E16.5 after IUEp at E14.5. D,F) Quantification of the proportion of PAX6+ mCherry+ (D) or TBR2+ mCherry+ (F) cells expressed in % over total mCherry+ cells; scale bar: 20 µm. Error bars indicate the variation of five Control and five DGCR8 OE electroporated cortices (s.e.m.); unpaired Student's t-test. *p-value < 0.05; **p-value < 0.01.
Fig 5: The DROSHA protein levels were elevated in alveolar macrophages during bleomycin-induced pulmonary fibrosis. (A) Representative immunohistochemistry image of DROSHA in lung tissues from wild-type (WT) mice were exposed to PBS or bleomycin via oropharyngeal aspiration. Positive area and cells are indicated by the black arrow. Scale bars, 200 µm. Results are representative of three independent experiments. (B) Representative immunoblot analysis for DROSHA and DGCR8 (left) and densitometry quantification of DROSHA and DGCR8 levels (normalized to levels of ß-actin) (right) in lung tissues from WT mice exposed to PBS (n = 3) or bleomycin (n = 5) via oropharyngeal aspiration. For immunoblots, ß-actin was used as loading control. Data are representative of three independent experiments. Data are mean ± SEM. * p <0.05; by Student’s two-tailed t-test. (C) Representative immunofluorescence image of F4/80 (Red), DROSHA (Green), and DAPI (Blue) staining in lung tissues from WT mice exposed to PBS or bleomycin via oropharyngeal aspiration. Positive area and cells are indicated by white arrows. Scale bars, 200 µm. Quantification of co-localization positive cells between DROSHA and F4/80 (the percent of co-localization positive cells in total 100 cells in 10 individual images per group) (right) in lung tissues from WT mice exposed to bleomycin (n = 5) or PBS (n = 3) via oropharyngeal aspiration. Data are mean ± SEM. ** p <0.01, * p <0.05; by Student’s two-tailed t-test.
Supplier Page from Abcam for Anti-DGCR8 antibody [EPR18757]