Effect of SCML2B on the activity of CDK2/CYCE/p21-p27 <i>in vitro</i>.

<p>(A–B) Kinetic analysis of the kinase activity of CDK2/CYCE/p27 or CDK2/CYCE/p27/SCML2B (A) and CDK2/CYCE/p21 or CDK2/CYCE/p21/SCML2B (B) complexes with increasing concentrations of histone H1e, with the products analyzed by Western blot using antibody specific to H1T146Ph (lower panel) and quantified by densitometric analysis of the bands from three different experiments (upper panel). The results were fitted to Michaelis–Menten kinetics, and the amount of histone H1e was in 2–20 molar excess of SCML2B.</p

Cellular distribution and composition of SCML2B-containing complexes.

<p>(A) Schematic representation of the two protein isoforms encoded by the <i>SCML2</i> gene in <i>Homo sapiens</i>. (B) Western blot analysis of nuclear and cytoplasmic extracts of K562 cells using antibodies specific for SCML2, EZH2, and tubulin (left). Western blot of cytoplasmic, nuclear extract, and solubilized nuclear pellet of HeLa cells using an antibody specific for SCML2 (right). (C) Immunofluorescence analysis of SCML2 in HeLa cells transfected with two different siRNAs specific for SCML2. DAPI staining is shown on the left, and staining with an SCML2-specific antibody is shown on the right. (D) Proteins identified by mass spectrometry in the fraction containing purified SCML2B, indicating number of peptides for each protein and coverage. (E) Fractionation of purified SCML2B was performed by size exclusion chromatography and analyzed by Western blot for SCML2, CYCE2, CYCB2, CDK2, p27, and USP3. Molecular weight markers are indicated on the left. (F) Immunoprecipitation of CDK2 and SCML2 from nuclear extracts from HeLa cells. Five percent of the input (In) is shown, along with the flow-through (FT) and elution (E) fractions. A nonspecific IgG pull-down is shown as control.</p

SCML2B stabilizes p21.

<p>(A) Western blot analysis of p21, p27, and actin, using whole cell extracts from U2OS cells treated with two different siRNAs (#1, #2) for SCML2 or a control siRNA (C) for 72 h, and then incubated in the presence of 5 µM MG132 or DMSO. (B) Quantification of the densitometric analysis for p21 and p27, from three different experiments performed as in (A). The intensity was corrected for the expression of actin, and the ratio of the levels after MG132 treatment relative to those of DMSO treatment is shown for each siRNA. *<i>p</i><0.01 (C) U2OS cells were transfected with a control siRNA and plasmid (Control), with an siRNA against SCML2 and a control plasmid (si#2) or an siRNA against SCML2 and a plasmid expressing SCML2B resistant to the siRNA (si#2+SCML2B) for 48 h. Cells growing asynchronously (top panel) or released from an arrest in mitosis (5 h, middle panel, and 8 h, bottom panel) were then incubated with 25 µg/ml cycloheximide, and whole cells extracts were analyzed at different time points by Western blot with antibodies specific for p21. Densitometric quantification of the levels of p21 from two different experiments, each of them loaded in duplicate, is shown. The data were fitted to a one phase decay equation.</p

SCML2 interacts with CDK2/CYCE2 complexes in G1 and regulates their activation.

<p>(A) Western blot analysis of the SCML2B pull-down in 293 cells expressing FS-SCML2B, from asynchronously growing cells (As), and cells treated with thymidine (Thy) or nocodazole (NCZ). The pull-down was analyzed using SCML2, CDK2, CYCE2, and BMI1 antibodies. Shown on the right is 1.5% of the input. (B) Immunoprecipitation of CDK2 in cells treated with a control siRNA (C) or with siRNA specific for SCML2 (si #2) or p21 (p21), arrested in mitosis with nocodazole and released for different times, as indicated. Two percent of the input is shown (left) along with the elution (right). (C) Densitometric analysis of the amount of SCML2 pulled down by CDK2 at different time points after exit from mitosis, as in (B); the percentage of pull-down was normalized versus the input material and related to the amount of CDK2 in the pull-down. (D) Quantification of the kinase activity of CYCE2-associated complexes pulled down from control-treated U2OS cells and cells treated with siRNA #2 for SCML2, either growing asynchronously (As) or at different times after release from arrest in mitosis with nocodazole, as indicated. The reaction was performed in replicates, and the experiment was repeated twice. The levels of H1T146Ph were measured by Western blot with specific antibodies, and the levels of CYCE2 and p21 in the reaction are also shown.</p

Role of SCML2 in cell cycle.

<p>(A) Western blot analysis of extracts prepared from U2OS cells treated with two different siRNAs (#1, #2) against SCML2 or a control siRNA (C), using antibodies specific to SCML2, CDK2, p27, and p21. (B) Cell-cycle distribution of U2OS cells after SCML2 knockdown using the siRNAs indicated, measured by PI staining and FACS. *<i>p</i><0.05, **<i>p</i><0.01. (C–D) Cell-cycle distribution of U2OS cells after treatment with a control siRNA (Control), siRNA against either SCML2 (#2) or p21, or a combination thereof for SCML2 and p21 (C), or for SCML2 (#2) and a combination of SCML2 and CYCE2 (D), measured by PI staining and FACS. Statistical significance was determined with paired <i>t</i> tests. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. (E) Western blot analysis of extracts prepared from U2OS cells treated with siRNA against either SCML2 (#2), or p21 or CYCE2, or a combination thereof for SCML2 and p21, or for SCML2 and CYCE2, or a control siRNA (first column), using antibodies specific to SCML2, CYCE2, p21, CYCB2, p27, and USP7 (loading control). (F) U2OS cells were synchronized in mitosis with nocodazole after SCML2 knockdown, and the cell-cycle distribution after 16 h of release is compared. (G) Quantification of the proportion of cells in S phase at different time points after release from mitotic arrest from the experiment shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001737#pbio.1001737.s008" target="_blank">Figure S8</a>. *<i>p</i><0.05.</p

Regulation of G1/S progression by SCML2.

<p>(A) Western blot analysis of nuclear extracts isolated from U2OS cells transfected with siRNA against either SCML2 (#2) or p21 (si p21) or a control siRNA (si C). Cells were collected every 2 h, as indicated, after release from a mitotic arrest induced with nocodazole. The extracts were analyzed using antibodies specific to USP7 (loading control), SCML2, CYCE2, CYCB2, CDK2, p27, and p21. (B) Densitometric analysis of the levels of p21 at different points after release from mitosis in cells treated with a control siRNA or specific for SCML2 (si #2). The expression was normalized to the levels of CDK2, and the mean of four different experiments is shown. (C) Western blot analysis of nuclear extracts isolated from U2OS cells transfected with a control siRNA and plasmid (Control), with an siRNA against SCML2 and a control plasmid (si #2) or an siRNA against SCML2 and a plasmid expressing SCML2B resistant to the siRNA (si #2+SCML2B). Cells were collected every 2 h, as indicated, after release from a mitotic arrest induced with nocodazole. The extracts were analyzed using antibodies specific to p21. Ponceau staining is shown as loading control. (D) Densitometric analysis of the levels of p21 at different time points after release from mitosis in two independent experiments.</p

ES cell differentiation modulates the interaction of SCML2 with CDK2.

<p>(A) Immunoprecipitation of CDK2 in nuclear extracts from control-treated H9 cells (C) or cells treated with 15 µM Nutlin for 3 d (Nut). The material pulled down was analyzed with specific antibodies for SCML2, CDK2, and p21. Immunoprecipitation with a nonspecific IgG is used as control, and 1% input is shown (In). (B) Western blot analysis of the levels of Nanog and p21 in nuclear extracts of control H9 cells (C) and cells treated with 15 µM Nutlin for 3 d (Nut). Ponceau staining is shown as loading control. (C) Model for the proposed mechanism of action of SCML2B on the regulation of G1 progression. SCML2B and p21 or p27 cooperatively bind and inhibit CDK2/CYCE complexes in early G1 (step I, left), preventing their premature activation. As p21 and p27 levels increase, their association with CDK2/CYCE becomes more stable and independent of SCML2B, and restricts progression into S phase (step II, middle). Over time, increasing amounts of CDK2/CYCE lead to the phosphorylation of p21 and p27 and promote their degradation to allow the entry into S phase (step III, middle). Our data show that in the absence of SCML2B step I is missing, which results in a less effective step II and an accelerated progression to step III.</p

Binding affinities of TDRD3 (aa 520–633), SMN (aa 80–170), and SPF30 (aa 65–150) to different methyl-arginine peptides in comparison with SND1 (aa 650–910).

<p>NB: No detectable binding.</p><p>*: data from Liu K et al, PNAS, 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030375#pone.0030375-Liu2" target="_blank">[20]</a>.</p

Surface representation of TDRD3 and SMN.

<p>(A) TDRD3 (B) SMN (PDB: 1MHN). The ligand is superimposed from the TDRD3-PG4 structure.</p

Fluorescence polarization binding curves of TDRD3 (aa 520–633), SMN (aa 80–170), and SPF30 (aa 65–150) to the PIWIL1-R4 peptides.

<p>The buffer used in the fluorescence polarization assay is 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM DTT and 0.01% Triton X-100. The data are measured at 25°C and corrected for background by subtracting the free-labeled peptide background. The Kd values are the average of three independent measurements.</p