1. Volume 39, Issue 3, Pages 373-384 (August 2010)
Smad Proteins Bind a Conserved RNA Sequence to Promote MicroRNA Maturation by Drosha 
Brandi N. Davis, Aaron C. Hilyard, Peter H. Nguyen, Giorgio Lagna, Akiko Hata 
Molecular Cell 
Volume 39, Issue 3, Pages (August 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2010 39, 373-384DOI: (10.1016/j.molcel.2010.07.011)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1
miRNA Array Analysis of TGFβ- or BMP-Regulated miRNAs in PASMC
(A) Unsupervised hierarchical clustering of the fold induction of microRNAs following 24 hr TGFβ or BMP4 treatment compared to mock treated PASMC was performed using Gene Pattern, and displayed as a heat map where red corresponds to induction of mature miRNA by growth factor treatment and green corresponds to reduction relative to the mock treated control.
(B) Sequence logo representing the conserved motif present in miRNAs in Cluster 1 is indicated. The overall height of each stack indicates the sequence conservation at that position (measured in bits). The x-axis represents the relative location of the conserved nt along the mature miRNA. For clarity, only 5 bp flanking the conserved CAGAC sequence are displayed.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
The R-SBE Sequence Is Essential for Smad-Mediated Regulation of Drosha Processing
(A) Schematic diagram of pre-miR-21 wild-type and mutant sequences. Red and blue characters indicate the mature miRNA sequence and the R-SBE sequence found in pre-miR-21, respectively. Yellow highlighted characters indicate mutations introduced.
(B) Mouse C3H10T1/2 cells were transfected with human pri-miR-21 expression constructs, followed by treatment with or without 3 nM BMP4 for 2 hr and subjected to qRT-PCR analysis using primers to detect exogenous pri-miR-21 or pre-miR-21, normalized to GAPDH. Fold induction by BMP relative to mock treated cells is displayed. Induction of WT, 5′ mut, and Loop mut by BMP4 is statistically significant (∗p < 0.05, n = 3).
(C) Cos7 cells were transfected with pri-miR-21 expression constructs along with Flag-Smad1, Flag-Drosha, or Flag-DGCR8 expression constructs, and stimulated with BMP4 for 2 hr. RNA-IP assay using anti-Flag antibody was performed, followed by PCR amplification of exogenous pri-miR-21. Relative enrichment above a nonspecific control IgG antibody IP control is shown. Expression of pri- or pre-miR-21 prior to IP was examined by qRT-PCR analysis using indicated primers, normalized to GAPDH (Input) (∗p < 0.05, n = 3).
Error bars represent SEM. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Introduction of R-SBE Is Sufficient for the TGFβ-Regulated Processing of Pri-miRNA
(A) Schematic of pre-miRNA sequence of cel-miR-84 wild-type and mutants. Red and blue characters indicate mature cel-miR-84 sequence and the location of the introduced R-SBE sequence, respectively.
(B) RNA-IP assay was performed in Cos7 cells transfected with Flag-Smad1 and different pri-cel-miR-84 constructs. Cells were treated with BMP4 for 2 hr followed by immunoprecipitation of RNA fragments with anti-Flag antibody, and PCR amplification of pri-cel-miR-84. Fold enrichment of Flag IP relative to IgG control is presented (∗p < 0.01, n = 3).
(C) Cos7 cells were transfected with pri-cel-miR-84 constructs or pri-miR-21 expression construct (positive control), followed by treatment with or without 3 nM BMP4 for 2 hr and subjected to qRT-PCR analysis using indicated primers, normalized to GAPDH. (∗p < 0.01, n = 3).
Error bars represent SEM. See also Figure S2.
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6. Figure 4 Direct Association of Smad MH1 Domain and the R-SBE
(A) In vitro transcribed wild-type pri-miR-21 was mixed with indicated recombinant, sepharose bead-immobilized GST-fusion proteins. Associated RNA was eluted, and subjected to qRT-PCR analysis to detect pri-miR-21. The relative amount of pri-miR-21 pulled down with GST-Smad fusion proteins, normalized to the amount pulled down with GST alone is presented (∗p < 0.01, n = 3).
(B) In vitro transcribed pri-miR-21 constructs were mixed with recombinant GST-Smad1(MH1) or GST alone and the relative amount of pri-miR-21 transcripts pulled down with GST-Smad1(MH1) fusion protein was normalized to the amount pulled down with GST alone and presented. Values labeled with the same letter do not differ significantly from one another (p < 0.05, n = 3).
(C) EMSAs were performed with 1 nM of radiolabeled pri-cel-miR-84(RSBE-Mid) (left panel) or the wild-type (WT) pri-cel-miR-84 (right panel) probe and recombinant GST-Smad1(FL, MH1 or MH2) proteins. Shifted bands as a result of specific binding to Smad1 are indicated. Asterisk indicates a nonspecific band. Experiment was performed 3 times and a representative blot shown.
(D) Schematic diagram of mature miRNA sequence of wild-type cel-miR-84, cel-miR-84 introduced with R-SBE (RSBE-Mid) and mutants (top panel). In vitro transcribed pri-cel-miR-84(RSBE-Mid), pri-miR-21 (positive controls), wild-type cel-miR-84 (negative control) or R-SBE mutants were mixed with recombinant GST-Smad1(MH1) or GST alone and the relative amount of pri-miRNAs pulled down with GST-Smad1(MH1) fusion protein was normalized to the amount pulled down with GST alone and presented (bottom panel). Values labeled with the same letter do not differ significantly from one another (p < 0.05, n = 3).
(E) C3H10T1/2 cells were transfected with pri-cel-miR-84 constructs (WT or mutants) or pri-miR-21 expression construct (control), followed by treatment with or without 3 nM BMP4 for 2 hr and subjected to qRT-PCR analysis using indicated primers, normalized to GAPDH (∗p < 0.01, n = 3).
(F) Synthetic RNA duplexes (miR-21 or cel-miR-67) were mixed with recombinant GST alone or indicated GST-Smad fusion proteins. Fold enrichment of RNA duplex pulled down with GST-Smad fusion proteins over GST protein alone is presented.
(G) Three- or 30-fold molar excess of synthetic DNA duplex, SBE1 or SBE2, or 30-fold molar excess of in vitro transcribed pri-miR-21 R-SBE mutant 3 (R-SBE-M3) were added during Smad1 pull-down assay using in vitro transcribed pri-miR-21(WT) conjugated to agarose beads. The amount of Smad1 associated with pri-miR-21 in the presence of competitors was examined by immunoblot analysis with anti-Smad1 antibody (top panel). The relative amount of Smad1 bound to pri-miR-21 was quantitated by densitometry and presented (bottom panel).
Error bars represent SEM. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Identification of miRNAs Regulated by the TGFβ Signaling Pathway Posttranscriptionally
(A) Sequence alignment of analyzed miRNAs containing R-SBE (left panel). Levels of expression of mature miRNAs, normalized to U6, were examined in PASMCs treated with 3 nM BMP4 or 400 pM TGFβ1 for 6 hr (right panel). miR-25 does not contain R-SBE and is not regulated by BMP4 or TGFβ1. Fold induction after treatment relative to mock treated PASMC is presented.
(B) PASMCs were pretreated with RNA pol II inhibitor α-amanitin for 5 hr followed by treatment with or without 3 nM BMP4 for 2 hr and subjected to qRT-PCR analysis using primers to detect pre-miRNAs or Id3, normalized to GAPDH. Fold change relative to untreated cells is presented (∗p < 0.05, compared to no treatment, n = 3).
(C) Human PASMCs, MDA-MB-468 cells, or U251 cells were treated with 3 nM BMP4 or 400 pM TGFβ1 for 2 hr and subjected to qRT-PCR using indicated primers, normalized to GAPDH. The fold induction relative to mock treated sample is presented.
(D) Time-course expression of indicated pri-miRs, normalized to GAPDH, was examined by qRT-PCR in PASMCs stimulated with 3 nM BMP4 (left panel) or 400 pM TGFβ1 (right panel) for 2 or 4 hr. Fold induction compared to untreated samples are presented.
Error bars represent SEM. See also Figure S4 and Table S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Smad Is Essential for Recruitment of Drosha
(A) RNA-IP assay was performed in PASMCs treated with BMP4 for 2 hr followed by immunoprecipitation of RNA fragments with anti-Smad1/5 antibody, anti-Drosha antibody, or nonspecific IgG (IgG), and PCR amplification was performed to detect indicated pri-miRNAs. Fold induction of binding relative to untreated PASMC is presented. Primers for miR-214 and -222 serve as negative controls because they are not regulated by TGFβ or BMP. A primer set recognizing the TMEM49 coding region (TM) was used as an additional negative control (∗p < 0.001, n = 3).
(B) PASMCs were transfected with nontargeting control siRNA (si-Control) or mixture of siRNAs for Smad1 and Smad5 (si-Smads). Twenty-four hours after transfection, cells were treated with 3 nM BMP4 for 2 hr and were subjected to RNA-IP analysis to examine recruitment of Drosha to indicated pri-miRNAs or TM using anti-Drosha antibody. The relative enrichment over IgG control IP is shown. qRT-PCR analysis of pri-miRNAs and pre-miRNAs, normalized to GAPDH, prior to immunoprecipitation is shown (Pre-miRNA and Pri-miRNA). As a control for R-smad knockdown, the level of Id3, normalized to GAPDH was monitored (∗p < 0.001, n = 3).
Error bars represent SEM. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions