To test whether the R-SMAD–p68–DROSHA complex assembles specifically on pri-miR-21, we performed an RNA-chromatin immunoprecipitation (ChIP) analysis on Cos7 cells co-transfected with pCMV-miR-21 and Flag-tagged SMAD1, SMAD3 or SMAD2. The association of SMAD1 (but not SMAD2 or SMAD3) with pri-miR-21 was induced threefold on BMP4 stimulation for 2 h ( and Supplementary Fig. 16a), whereas TGF-β increased binding to pri-miR-21 by SMAD2 and SMAD3, but not by SMAD1, indicating that the association between R-SMADs and pri-miR-21 is specifically regulated by ligand stimulation ().
Association of SMADs with pri-miRNA promotes processing by DROSHA
R-SMADs also interacted in a ligand-specific manner with pri-miR-21 in PASMCs (), whereas p68 constitutively associated with pri-miR-21 and the recruitment of DROSHA was moderately enhanced by either TGF-β or BMP4 (). Similar results were obtained for miR-199a (). The significant increase (P < 0.05) we observed in the association of DROSHA with pri-miR-21 and pri-miR-199a () suggests that binding of SMADs to the pri-miRNA might stabilize the association between DROSHA and the pri-miRNA. We detected a constitutive association of pri-miR-214 with p68 and DROSHA, but no interaction with SMADs (), confirming that pre-miR-214 is not regulated by BMP or TGF-β signals (Supplementary Fig. 17). Thus, recruitment of SMADs to the p68–DROSHA complex is pri-miRNA-specific.
A SMAD1 mutant that was non-phosphorylatable on BMP stimulation (SMAD1(3SA)) retained the ability to interact with pri-miR-21 (Supplementary Fig. 16a). Furthermore, bacterially expressed unphosphorylated GST–SMAD fusion proteins are able to interact with p68 (Supplementary Figs 14 and 15), indicating that receptor-mediated phosphorylation of R-SMADs is not essential for the association with pri-miRNA and suggesting that BMPs may affect the association between SMAD1 and pri-miRNAs primarily by controlling SMAD nuclear localization.
Pull-down experiments using partially purified GST–SMAD fusion proteins as bait confirmed that SMAD1, SMAD3 and SMAD5 can interact with pri-miR-21. Interestingly, both the MH1 and the MH2 domains of SMAD1 bound to pri-miR-21 (Supplementary Fig. 18). Because MH1 does not interact with p68 (Supplementary Fig. 14b), it is possible that MH1 interacts either with pri-miR-21 directly or with other miR-21-binding proteins.
In summary, BMPs and TGF-β stimulate the expression of a specific subset of miRNAs by inducing the formation of a complex comprising R-SMAD proteins, pri-miRNAs and subunits of the microprocessor complex such as DROSHA and p68.
Finally, we examined the possibility that ligand treatment may facilitate DROSHA-mediated production of pre-miRNA. In vitro pri-miRNA processing assays were performed by incubating radio-labelled pri-miR-21 substrate (480 nucleotides) with nuclear extracts from Cos7 cells treated with vehicle, BMP4 or TGF-β. Ligand treatment resulted in ~25% increase (BMP4, 28.5% ± 1.9% (mean ± s.e.m.; TGF-β, 24.2% ± 1.4%; triplicate experiments) in the production of a 72-nucleotide product corresponding to pre-miR-21, compared to incubation with extracts from mock-treated cells (). This result suggests that ligand-induced association of SMADs with the DROSHA complex increases pri-miR-21 cropping into pre-miRNA.