Smad promotes miRNA maturation as a component of the Drosha microprocessor. Brandi N. Davis 1 , Giorgio Lagna Ph.D 2 , & Akiko Hata, Ph.D 1,2 1. Biochemistry Department, Sackler School of Biomedical Sciences, Tufts University School of Medicine 2. Molecular Cardiology Research Institute, Tufts Medical Center, Boston, MA, USA 02111. You institution’s name here Abstract Results Conclusions Expression of microRNA (miRNA) is temporally and spatially regulated, however, the mechanism of miRNA regulation is largely unknown. In addition to transcriptional regulation of the pri-miRNA, the miRNA processing steps catalyzed by the Drosha and Dicer complexes are likely to be regulated. Indeed, we have found the pri-miRNA to pre-miRNA processing step is regulated by the Transforming Growth Factor β (TGFβ) family of growth factors. The TGFβ family of ligands transmits signals which are critical for both development and the maintenance of normal adult homeostasis. The signal transducers of TGFβ signaling, the Smads, are known as transcription factors which upon nuclear translocation bind target promoters and regulate gene expression. Here we demonstrate a novel role of smads in miRNA processing. Stimulation of the TGFβ/BMP signaling pathways results in an up-regulation of mature miR-21 without affecting the expression level of pri-miR-21. Instead, the result of Drosha processing, the pre-miR-21, is dramatically up-regulated by ligand stimulation. Smad is required for this processing step as knockdown of Smad by siRNA blocks induction of both the mature miR-21 and pre-miR-21. We demonstrate that Smad is recruited to, and promotes the processing of pri-miR-21 in a ligand-dependent fashion. Furthermore, we demonstrate that Smad is incorporated into the Drosha microprocessor complex through interaction with the RNA helicase p68 (also known as DDX5). In conclusion, our results demonstrate a novel mechanism of regulation of miRNA biogenesis by Smad. This is the first report of growth factor signaling-dependent regulation of miRNA maturation. Introduction miR21 is required for BMP-mediated SMC differentiation. A). PASMC were transfected with anti-miRs to reduce mature miR level ~75-90%. Following 24H vehicle or BMP4 treatment, mRNA level was quantitated by RT-PCR. Transfection with anti-miR21 resulted in a decrease in both the basal and induced level of ASMA. B). PASMC were treated as in a. and the level of ASMA protein determined by IF. Anti-miR21 dramatically inhibits BMP-mediated SMC differentiation. Furthermore, exogenous up-regulation of miR21 promotes SMC differentiation (data not shown). Together, these results indicate miR21 is required for the BMP-mediated differentiation of smooth muscle cells. Other miRs identified as highly expressed in SMC show similar (miR125) or opposite effects (miR221, miR100) on SMC differentiation and are currently under investigation. To more completely understand the effect of miR21 in smooth muscle cells, we screened both predicted and validated targets of miR21 in primary SMC. C.) The levels of Programmed Cell Death Protein 4 (PDCD4), a validated target of miR21, were measured by RT-PCR following transfection of indicated antimiRs. In both the controls, treatment of PASMC with BMP4 causes a reduction of PDCD4 mRNA, corresponding to the increase in mature miR21 following BMP4 treatment. This effect is blocked by transfection of anti-miR21, further confirming PDCD4 as a target of miR21. D). Up-regulation of PDCD4 by transient transfection in 10T1/2 cells was used to mimic anti-miR21. This treatment decreases both the basal and induced level of SMC markers, confirming PDCD4 as a functional target of miR21 in SMC. a. b. Smooth muscle cells are unique in their ability to undergo phenotypic modulation from a synthetic, proliferative state to a contractile, differentiated phenotype (a). Deregulation of this switching underlies many vascular disorders including hypertension, restenosis, and atherosclerosis. A variety of signaling pathways have been implicated in the control of smooth muscle differentiation. In particular, BMP and TGF- β have been shown to promote and maintain a differentiated SMC phenotype. The canonical TGF β super family signaling cascade is characterized by ligand binding to trans-membrane receptor kinases and subsequent activation of the smad proteins. Upon phosphorylation, the smads oligomerize and translocate into the nucleus to alter gene expression (b). TGF β signaling has been widely implicated in the control of both physiological and pathological response. Identification of miR expressed in differentiated SMC. A microRNA cloning approach was utilized to identify miR which are highly expressed in smooth muscle cells following BMP treatment to promote differentiation. To confirm the cloning results, Taq-man RT-PCR was performed to analyze the mature miR levels of human Primary Pulmonary Artery Smooth Muscle cells (PASMC) following 24H BMP treatment. Two miRs: miR21 and miR199a were strongly induced by BMP treatment compared to vehicle alone. c. d. b. a. BMP/TGF- ß promote drosha mediated processing of miR21 . A). qRT-PCR of PASMC treated with BMP4 for indicated times shows dramatic induction of pre- and mature miR21, but no change in the level of miR21 primary transcript. B). To confirm miR21 induction is due to a post-transcriptional event, PASMC were treated with α -amanitin prior to 2H BMP4 treatment. Despite block of transcriptional response (ID-1), miR21 induction is unaffected. C). In order to examine miR21 induction outside of the genomic context, human miR21 plasmid was transfected into mouse 10T1/2 cells and the level of exogenous pri- and pre-miR21 measured by RT-PCR following 2H BMP4 treatment. While the level of pri-miR21 transcript is unaltered by BMP treatment, pre-miR21 is strongly induced. D). In-vitro transcribed pri-miR21 was applied to nuclear extracts from mock, BMP, or TGF- ß treated cos7 cells. Addition of either ligand increased the degree of processing relative to mock by ~30%. Together, these results provide support for a novel pathway of microRNA processing regulation. a. b. d. c. Smads associate with the Drosha microprocessor. A). PASMC were transfected with siRNA against receptor smads 1 & 5 and the level of miR21 intermediates were measured by RT-PCR following 2H BMP4 treatment. Surprisingly, although transcription is not required for miR21 induction, down-regulation of smads abolished BMP-mediated induction of both pre- and mature miR21. This result indicates smads are required for BMP-mediated induction of miR21. B). Endogenous Drosha, and a sub-unit of the Drosha microprocessor, p68, were observed to co-immunoprecipitate with smad1 following BMP treatment of PASM. Indicating that smads are present within the Drosha processing complex. C). To further confirm the presence of smads within the drosha processor, RNA-chIP experiments were performed in PASM. Both miR21 and miR199a show a ligand-dependent induction of smad binding to the miR precursor. As a control, the smad occupancy of miR214 was examined. This miR is unaffected by BMP signaling and did not show binding of smads. Together these results indicate the nuclear accumulation of smads in response to ligand stimulation promotes association with the processing complex and the subsequent increase in pre-miR21. Mechanistically, smads may serve to promote processing of a subset of microRNAs by recruitment of the drosha processor to pri-miRs, stabilization of the processing components, or alteration of the enzymatic activity of Drosha. Pri-miR Pre-miR mature-miR21 SMC differentiation PDCD4
Down-regulation of PDCD4 by miR21 is required for SMC differentiation in response to BMP.
BMP and TGF- β promote the drosha processing of a subset of microRNAs
Receptor smads are a novel subunit of the drosha microprocessor complex
Investigate other functional targets of miR21 in SMC
Determine the mechanism of smad association with Drosha
Investigate other smad-regulated miRs
c. a. b. Bernhard Schmierer & Caroline S. Hill December 2007 doi:10.1038/nrm2297 Owens et al. Physiol. Rev. 84: 767-801, 2004 Synthetic Contractile miR BMP/TGF- β