Fig 2: All Drosha splice variants form the microprocessor complex with DGCR8 and bind additional interaction partners. (A) Schematic representations of the microprocessor complex (i) and an additional Drosha-containing complex (ii). A selection of interacting partners is represented. Additional proteins are indicated with dashed lines. (B) Immunoprecipitation of Flag-tagged DGCR8 pulled down its interaction partner V5-tagged Drosha. (C) Immunoprecipitation of Flag-tagged Drosha pulled down its HA-tagged microprocessor co-factor DGCR8. (D) Immunoprecipitation of YFP-tagged Drosha pulled down endogenous DGCR8, (E) endogenous EWS and p68 and (F) exogenous p68. All co-immunoprecipitation experiments were performed from cell lysates of HEK293 cells transiently transfected in at least two biological replicates with the respective Drosha splice variant (YFP-Drosha-V5) construct as well as Flag/HA-DGCR8 or Flag-AGO2 (B), HA-DGCR8 (C) or p68 (F), respectively.

Fig 3: TTP negatively regulates the levels of Lin28a in human cancer cells. (A–D) Overexpression of TTP inhibits Lin28a levels in PA1 cells. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 for 24 h. (A) The levels of TTP, Drosha, Dicer, Ago2 and Lin28a proteins were determined by western blot assays. (B) The expression levels of TTP and Lin28a were determined by semi-qRT-PCR. (C) The level of Lin28a was determined by qRT-PCR. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01). (D) Overexpression of TTP decreases Lin28b levels in PA1 cells. The level of TTP and Lin28b were determined by RT-PCR (top panel) and western blot (bottom panel). (E–I) Downregulation of TTP by siRNA increases Lin28a levels and decreases let-7b in HCT116 cells. HCT116 cells were transfected with TTP-specific (TTP-siRNA) or scRNA. After 24 h, the levels of TTP and Lin28a were determined by qRT-PCR (E and F) and western blot assays (G). The level of let-7b was determined by qRT-PCR (H) and cell viability was assessed by measuring absorbance at 490 nm using a MTS cell proliferation assay (I). The levels obtained from mock-transfected HCT116 cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01; ***P < 0.001). ns, not significant. (J) The level of TTP protein is inversely correlated with those of Lin28a protein within several human cell lines. Levels of TTP and Lin28a proteins were determined by western blot analysis in PA1 (ovarian teratocarcinoma), MCF7 (breast adenocarcinoma), AGS (gastric adenocarcinoma), K562 (erythroleukemia), HepG2 (hepatocellular carcinoma) and NT2 (neuronally committed teratocarcinoma) cells. β-Actin was detected as the loading control. (K) TTP expression level is inversely correlated with that of Lin28a in human ovarian tissues. Representative TTP and Lin28a immunohistochemical staining in normal and ovarian adenocarcinoma tissues. Normal ovarian tissues showed strong immunoreactivity for TTP, where as the ovarian adenocarcinoma showed strong positive staining for Lin28a.

Fig 4: A TLX peptide promotes miR-219 processing.(a) Mapping p68 and Drosha-interacting domain in TLX. A schematic of TLX deletion mutants and the Drosha/p68-interacting domain (Dpi) is shown on the left. A summary of p68 and Drosha-binding results is shown on the right. (b) Deletion of TLX residues 340–359 reduced the interaction of TLX with p68 substantially. HEK293T cells were transfected with HA-tagged full-length TLX (residues 1–385) or its deletion mutants (residues 1–306, 1–340 or 1–359). Lysates were immunoprecipitated (IP) with HA antibody (αHA), then probed with p68 antibody (αp68) in western blot analysis (WB). (c) Deletion of TLX residues 340–359 reduced TLX interaction with Drosha. HEK293T cells were transfected with Flag-tagged Drosha and HA-tagged full length or deletion mutants of TLX. Lysates were IP with Flag antibody (αFlag), then probed with HA antibody (αHA). A nonspecific (ns) band in the western blot was indicated. (d,e) Expressing the Dpi peptide abolished the interaction of TLX with Drosha (d), but not the interaction of TLX with HDAC5 (e), as revealed by co-IP analysis. An empty vector (−) and a control peptide (C) were included as negative controls for the Dpi peptide. Cell lysates were IP with anti-Flag antibody, then blotted with anti-HA or anti-Flag antibody. The expression of individual proteins in the transfected cells was shown by immunoblotting as input. (f) Expression of the Dpi peptide promotes miR-219 processing. miR-219 processing was monitored using the miR-219-Glo reporter. Expressing the Dpi peptide decreased miR-219-Glo activity compared to expressing the empty vector (−) or a control peptide (C); n=3. ***P<0.001 by Student's t-test. (g–i) The levels of pre-miR-219 (h) and mature miR-219 (i), but not pri-miR-219 (g), were increased by expressing the Dpi peptide, as revealed by RT–PCR. n=4 (g); n=4 (h); n=3 (i). *P<0.05 and **P<0.01 by Student's t-test in panels (h,i). ‘n' represents experimental repeats in panels (f–i).

Fig 5: GSK3β activation enhances MiR biogenesis and GSK3β modulation alters pre-miR synthesis (A) qRT-PCR analysis of miR-27a, miR-23a, miR-24, miR-141 and miR-182 levels in HEK293T cells transfected with pMT23-HA-GSK3β(S9A) for 48 h. U18 was used as a normalisation gene. (B) qRT-PCR analysis of (i) pri-miR-23a27a24-2, (ii) pri-miR-141/200c and (iii) pri-miR-182 expression from HEK293T cells transfected with pMT23-HA-GSK3β(S9A) for 48 h. β-Actin was used as normalisation gene. (A and B) Columns: mean ± SEM for three independent experiments performed in triplicate. (C) Western blot analysis of Drosha, AGO2, FUS, DGCR8 and p72 protein levels in HEK293T cells transfected with pMT23-HA-GSK3β(S9A) for 48 h. β-Tubulin was used as a loading control. (D) Luciferase activity in extracts of COS-1 cells transfected with 250 ng CMV-Pri-miR-23a27a24-2-GL4 and 0, 100 or 400 ng pMT23-HA-GSK3β(S9A) for 48 h. Luciferase was normalised for transfection efficiency (β-galactosidase activity) and mean ± SEM of three independent experiments performed in duplicate is shown. (E) In vitro Drosha-mediated pri-miR processing assay analysis of pre-miR levels following incubation of in vitro transcribed radio-labelled pri-miR-23a27a24-2 with Microprocessor complex immunoprecipitated from HEK293T cells transfected with Flag-Drosha ± GSK3β-WT, -S9A or -K85R for 48 h. Products of in vitro processing reactions were resolved on a 6% acrylamide:urea gel and exposed to film. A representative image of (i) three independent experiments is shown. Densitometry was performed using Image J software (ii and iii). Columns: mean ± SEM for three independent experiments. Images of biological replicate experiments and complete gel images can be found in Supplementary Figure S2B and S3. (F) qRT-PCR analysis of (i) pre-miR-27a and (ii) pri-miR-23a27a24-2 levels in HEK293T cells treated with either 2 μM 99021, an equal volume of DMSO or transfected with pMT23-HA-GSK3β(WT) pMT23-HA-GSK3β(S9A) or pMT23-HA-GSK3β(K85R) for 48 h. L19 was used as a normalisation gene. Columns: mean ± SEM for three independent experiments performed in duplicate. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0001. See also Supplementary Figures S2 and S3.