1. Volume 24, Issue 1, Pages 13-25 (January 2013)
piRNA-Triggered MIWI Ubiquitination and Removal by APC/C in Late Spermatogenesis 
Shuang Zhao, Lan-Tao Gou, Man Zhang, Li-Dong Zu, Min-Min Hua, Ye Hua, Hui-Juan Shi, Yong Li, Jinsong Li, Dangsheng Li, En-Duo Wang, Mo-Fang Liu 
Developmental Cell 
Volume 24, Issue 1, Pages (January 2013)
DOI: /j.devcel
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2. Developmental Cell 2013 24, 13-25DOI: (10.1016/j.devcel.2012.12.006)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 MIWI Interacts with APC/C
(A) Co-IP assays of the interactions between APC/C subunits and Flag-MIWI in 293T cells cotransfected with piR-1 (lanes 6–8) or control Scr siR (lanes 3–5). Flag-MIWI cotransfected with Scr siR (lanes 3 and 6), APC7 siR (lanes 4 and 7), or APC10 siR (lanes 5 and 8) were IPed by anti-Flag antibody. Top: anti-Flag IP pellets immunoblotted with anti-MIWI (panel 1, lanes 3–8; as a loading control) or indicated anti-APC/C subunit antibodies (panels 2–6, lanes 3–8). The IgG IP pellet (lane 1) and lysate of Flag-MIWI transfected-293T cells (lane 2) served as negative and positive controls, respectively. Bottom: immunoblotting of cell lysates for Flag-MIWI or indicated APC/C subunits, with β-actin serving as a loading control. Error bar is given as the SD of three independent experiments.
(B and C) Co-IP assays of the interactions between endogenous MIWI and APC/C subunits in adult mouse testis (B) or isolated mouse male germ cells (C). (B) IB of MIWI in indicated anti-APC/C subunit IP pellets (lane 2). IB of MIWI in testis lysate (lane 1) or IgG IP pellet (lane 3) served as positive and negative controls, respectively. (C) IB of indicated APC/C subunits in anti-MIWI IP pellets from SC (lane 1), RS (lane 3), and LS (lane 5). IgG IP pellets served as negative controls (lanes 2, 4, and 6). Results shown are representative of three independent experiments.
See also Figure S1.
Developmental Cell  , 13-25DOI: ( /j.devcel )
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4. Figure 2
MIWI Is a Substrate of APC/C, and MIWI Ubiquitination by APC/C Requires piRNAs
(A) The D-box element is conserved in vertebrate PIWI proteins.
(B) Detection of MIWI ubiquitination in 293T cells cotransfected with p3 × Flag-MIWI and pEF-HA-Ub (lane 2), and mouse testicular piRNAs (lane 3), piR-1 (lane 4), or control Scr siR (lane 5). Top: anti-Flag IP pellets immunoblotted with anti-Ub for Ub-MIWI conjugates (upper), or anti-MIWI to normalize the loading amounts (lower). Bottom: IB of MIWI protein levels in respective cell lysates, with β-actin serving as a loading control. See also Figures S2A and S2B.
(C) Detection of MIWI ubiquitination in 293T cells cotransfected with p3 × Flag-MIWI, pEF-HA-Ub, piR-1, and wild-type Emi1 (lane 2) or a deficient Emi1 mutant (S145/149A) (lane 3).
(D) Detection of MIWI ubiquitination in 293T cells cotransfected with p3 × Flag-MIWI, pEF-HA-Ub, piR-1, and control Scr siR (lane 1) or siRNAs against APC2 (lane 2), APC7 (lane 3), and APC10 (lane 4), respectively. Top: detection of Ub-MIWI conjugates, using procedures similar to that described in (B). Bottom: IB analysis of APC2, APC7, APC10, and MIWI proteins in respective cell lysates, with β-actin serving as a loading control. See also Figure S2C.
(E) Detection of MIWI ubiquitination in p3 × Flag-MIWI, pEF-HA-Ub, and piR-1 cotransfected HeLa cells synchronized in M phase (lane 1) or S phase (lane 2). Ub-MIWI conjugates were detected as described in (B).
(F) Detection of ubiquitination of wild-type MIWI (lane 1) and a piRNA-loading-deficient MIWI mutant (Y346/347A) (lane 2) in 293T cells. See also Figures S2D and S3D.
(G) APC/C-mediated ubiquitination of MIWI in vitro. The reactions were carried out in the absence of RNA oligonucleotides (lane 3), or in the presence of piR-1 (lane 4) or control Scr siR (lane 5). IgG IPed beads (lane 1) or omission of MIWI (lane 2) in the reactions served as negative controls. See also Figures S2E and S2F.
(H) Effect of piR-1 on Flag-MIWI expression in 293T cells. Top: IB of Flag-MIWI protein level (left) and quantitative RT-PCR analysis of MIWI mRNA level (right) in Scr siR- (lane 1) or piR-1-transfected cells (lane 2). Bottom: effect of MG132 on piRNA-induced MIWI degradation.
Results shown in (B)–(H) are representative of three independent experiments. Error bar is the SD of three independent experiments.
Developmental Cell  , 13-25DOI: ( /j.devcel )
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5. Figure 3
Screening Amino Acid Residues Critical for MIWI Ubiquitination
(A) Schematic illustrations of MIWI mutants.
(B and C) Ubiquitination of MIWI K→A (B) or K→R mutants (C) in 293T cells. (B) Lanes 3–9: D-box mutant and indicated K→A mutants in the presence of piR-1, using wild-type MIWI in the absence (lane 1) or presence of piR-1 (lane 2) as negative or positive controls. (C) Lanes 2–7: Indicated K→R mutants in the presence of piR-1, using wild-type MIWI in the presence of piR-1 as a positive control (lane 1). The experiments were conducted as described in Figure 2B. Results shown in (B) and (C) are representative of three independent experiments. See also Figure S3.
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6. Figure 4
piRNA Loading Induces a Conformational Change of MIWI and Enhances MIWI Binding to the Substrate-Binding APC10
(A) Co-IP assays of the interaction between Myc-APC10 and Flag-MIWI in 293T cells (lane 1), or in the presence of Scr siR (lane 2) or piR-1 (lane 3). Top: anti-Myc IP pellets immunoblotted by anti-Myc or anti-Flag antibodies. The relative efficiency of MIWI co-IP (anti-Flag/anti-Myc) is indicated in parentheses (the one without RNA transfection [lane 1] is set as 100%). Bottom: cell lysates immunoblotted by anti-Myc or anti-Flag antibodies, with β-actin serving as a loading control.
(B) Co-IP assays of the interaction between Myc-APC10 and indicated MIWI mutants in 293T cells cotransfected with piR-1 (lanes 2, 4, 6, and 8) or control Scr siR (lanes 1, 3, 5, and 7). Procedures are similar to that in (A).
(C) Intrinsic tryptophan fluorescence assays of purified wild-type MIWI (left) and Y346/347A mutant (middle) in the absence or presence of piRNAs. Fluorescence intensities (in arbitrary units [a.u.]; y axis) of MIWI (—), MIWI mixed with piR-1 (—), or control Scr siR (⋅⋅⋅⋅) were measured using excitation at 283 nm and emission between 300 and 500 nm (x axis). piR-1 (-⋅-) and Scr siR (-⋅-) without MIWI served as negative controls. Right: Coomassie blue staining of proteins purified from 293T cells transfected with control p3 × Flag vector (lane 1), wild-type Flag-MIWI (lane 2), or Y346/347A (lane 3). Results shown are representative of three independent experiments. Error bar is the SD of three independent experiments.
Developmental Cell  , 13-25DOI: ( /j.devcel )
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7. Figure 5
Ubiquitination of MIWI In Vivo Occurs Only in Adult Testes and LS
(A) MIWI ubiquitination in mouse testes at different time points. Top: detection of MIWI ubiquitination in 7-dpp (lane 1), 14-dpp (lane 2), 20-dpp (lane 3), 5-week (lane 4), and 8-week testes (lane 5). Top: anti-MIWI IP pellets immunoblotted with anti-Ub for Ub-MIWI conjugates (top) or anti-MIWI to normalize the loading amounts (bottom). Bottom: RNA co-IP assays of MIWI associated-piRNAs (upper) in 7-dpp (lane 1), 14-dpp (lane 3), 20-dpp (lane 5), and 5-week (lane 7) testes (top), with IgG-IPed RNA as the negative controls (lanes 2, 4, 6, and 8) and anti-MIWI IB as loading references (bottom). See also Figures S4A and S4B.
(B) MIWI ubiquitination in germ cells. Top: detection of MIWI ubiquitination in SC (lane 1), RS (lane 2), and LS (lane 3). Bottom: RNA co-IP assays of MIWI associated-piRNAs in SC (lane 1), RS (lane 3), and LS (lane 5). The procedures are similar to that described in (A). See also Figures S4C–S4E.
(C) Double immunostaining of MIWI (red) and APC10 (green) using adult testis sections (top) or isolated mouse male germ cells (bottom). Nuclei were stained with DAPI (blue). Colocalization of MIWI and APC10 is shown in yellow. Scale bar: 100 μm in sections and 10 μm in cells.
(D) Co-IP assays of the interaction between MIWI and APC10 in SC (lane 1), RS (lane 2), and LS (lane 3) isolated from adult testes. Top: anti-APC10 IP pellets immunoblotted by anti-MIWI, with anti-APC10 IB as a loading control. Bottom: cell lysates immunoblotted by anti-MIWI or anti-APC10 antibodies, with β-actin serving as a loading control. Results shown are representative of three independent experiments.
Developmental Cell  , 13-25DOI: ( /j.devcel )
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8. Figure 6
MIWI and piRNAs Are Coordinately Eliminated in Late Spermatids
(A) Immunostaining of LS from testes transduced by shAPC10:GFP (I and III) or pSilencer:GFP (II and IV) with anti-MIWI (I and II, red) and anti-APC10 (III and IV, red). Representative staining images are shown on the left, and quantification of anti-MIWI staining fluorescent densities is shown at the top right (mean ± SD, n = 100 per group). See also Figures S5A–S5F.
(B) Effect of APC10 knockdown on MIWI ubiquitination in LS. GFP+ LS from testes transduced by shAPC10:GFP (lane 2) or pSilencer:GFP (lane 4), with respective GFP- cells from shAPC10:GFP (lane 1) or pSilencer:GFP (lane 3) transduced testes as controls. Top: anti-MIWI IP pellets immunoblotted with anti-Ub for Ub-MIWI conjugates (upper) or with anti-MIWI to normalize the loading amounts (lower). Bottom: IB of MIWI and APC10 in respective LS, with β-actin serving as a loading control.
(C) Immunostaining of LS from testes transduced by wild-type MIWI-IRES-GFP (I and III) or mutant Y346/347A-IRES-GFP (II and IV) with anti-Flag (I and II, red) and anti-MIWI (III and IV, red). Representative staining images are shown on the left, and quantification of anti-MIWI staining fluorescent densities is shown at the top right (mean ± SD, n = 100 per group). See also Figure S6.
(D and E) FISH assays of piRNAs (red) in LS from testes transduced by shAPC10:GFP (D, top), pSilencer:GFP (D, bottom), Flag-MIWI-IRES-GFP (E, top), or Y346/347A-IRES-GFP (E, bottom), respectively. Representative FISH images are shown on the left, and quantification of FISH fluorescent densities is shown on the right (mean ± SD, n = 100 per group).
(F) PCR analyses for GFP DNA sequences in genomic DNA from LS (top) and epididymal sperm (bottom) isolated from mice transduced with shAPC10:GFP (lanes 4–8), pSilencer:GFP (lanes 9–13), MIWI-IRES-GFP (lanes 14–18), or Y346/347A-IRES-GFP (lanes 19–23), respectively. Genomic DNA samples from wild-type background (lanes 2 and 25) and Oct4-GFP transgenic mice (lanes 3 and 24) served as negative and positive controls, respectively.
(G) Model for coordinated elimination of MIWI and piRNAs in late stages of spermatogenesis. MIWI protein in SC, RS, and LS is shown in red.
GFP images (green) and arrowheads in (A), (C), (D), and (E) indicate cells that were successfully transduced with the constructs. Nuclei were stained with DAPI (blue); scale bar: 10 μm. Results shown in (A)–(F) are representative of three independent experiments.
Developmental Cell  , 13-25DOI: ( /j.devcel )
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