Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 1. Nucleolar SIRT7 participates in multiple pathways in ribosomal biogenesis. A, B, HeLa cells treated with RNase A or buffer alone (Ctrl). Localizations of endogenous MYBBP1A, SIRT7, and RPA194 were detected via immunofluorescence microscopy. C, SIRT7 interacts with proteins involved in ribosome biogenesis. Rela- tive abundances of SIRT7-interacting proteins specific to ribosome biogenesis are indicated by the logarithm of the ratio of NSAF/PAX values. Node color and size correlate with NSAF/PAX ratios. Protein networks were assembled by STRING and visualized by Cytoscape. D, E, representative collision-induced dissociation MS/MS spectra of doubly charged tryptic peptides from mTOR (D) and GTF3C1 (E). Assigned b and y ion sequence fragments and prominent neutral losses from the precursor are labeled. BPI, base peak intensity.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Immunofluorescence, Microscopy, Tandem Mass Spectroscopy, Sequencing, Labeling

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 2. SIRT7 knockdown negatively regulates synthesis rate of rRNA. A, transcription inhibition in mitosis phase. Fluorescence mi- croscopy shows EU incorporation (EU-Alexan 488) into RNA 1 h after pulse-labeling. Cells undergoing mitosis exhibit chromosome con- densation (arrows). B, protein levels monitored via Western blotting following siRNA treatment (2 days). C, reduced synthesis rates of nascent RNA upon SIRT7 knockdown. Top: selected fluorescent images from EU-labeled HeLa cells treated with siRNA for 2 days. Relative EU levels, shown in parentheses, were calculated by normal- ization to the mean fluorescence value of control siRNA-treated cells. “Cell #” indicates the number of cells measured for each experiment. Bottom: normalized total cell fluorescence output from cells treated with siRNA targeting SIRT7. Mean fluorescence value (m) and stand- ard deviations (S.D.) for each condition are as follows: ctrl, m 69k, S.D. 44k; SIRT7, m 34k, S.D. 21k. p values were calculated using Student’s t test. Images were acquired using a 60 oil immer- sion lens; bar, 10 m. D, EU fluorescence distribution following pulse- labeling in cells treated with siRNA (si), with or without RNaseA, as analyzed via flow cytometry. E, pulse-and-chase experiment for measuring RNA stability. HeLa cells were treated with control or SIRT7 siRNA for 2 days and then EU labeled for 1.5 h. Then the cells were chased with different time points.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Knockdown, Inhibition, Fluorescence, Labeling, Western Blot, Control, Flow Cytometry

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 3. SIRT7 associates with ribosome units and regulates protein synthesis rate. A, co-localization of SIRT7-EGFP and RPL11 within nucleoli. A cell line stably expressing SIRT7-EGFP fusion pro- tein was transfected with RPL11 cDNA and monitored via immuno- fluorescence microscopy with a 60 oil immersion lens; bar, 10 m. B, co-fractionation of RPL5, RPL11, and SIRT7. Cells were treated with control and SIRT7 siRNA for 2 days. The distributions of ribo- some proteins and SIRT7 were analyzed by sucrose gradient. Pro- teins from individual fractions were precipitated and analyzed via Western blotting. C, SIRT7 knockdown inhibits protein synthesis. Protein synthesis rates after siRNA treatment for 3 days were moni- tored via pulse L-homopropargylglycine labeling followed by Click-iT reaction with a fluorescent dye.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Stable Transfection, Expressing, Transfection, Fluorescence, Microscopy, Fractionation, Control, Western Blot, Knockdown, Labeling

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 4. SIRT7 knockdown preferentially suppresses protein syn- thesis rates and inhibits proliferation. A, preferential inhibition of protein synthesis by SIRT7. Top: comparison of RNA and protein synthesis rates following siRNA treatment for 2 days. Bottom: knock- down efficiencies of individual siRNA were monitored via Western blotting. B, HeLa cells were transfected with SIRT7 siRNA and differ- ent plasmids (GFP, wild type, 111) for 2 days. The protein synthesis rates were measured using an HPG labeling method (top panel). The levels of SIRT7 were confirmed by Western blotting (bottom panel). WT: SIRT7 wild type; S111A: SIRT7 deacetylation-impacted mutant. GFP served as a control for overexpression. C, after siRNA treatment for 2 days, HeLa cells were treated with nucleotide releasing buffer for the detection of ATP levels. ADP levels were sequentially measured by adding ADP converting enzyme. The ratio of ADP to ATP in each condition was calculated. Camptothecin (CPT) was used as a control. D, E, cell proliferation rates following siRNA treatments for 2 and 3 days, respectively. p values were calculated using Student’s t test.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Knockdown, Inhibition, Comparison, Western Blot, Transfection, Labeling, Mutagenesis, Control, Over Expression

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 5. SIRT7 regulates the functions of mTOR and Pol III com- plexes. A, validation of SIRT7 interaction by reciprocal immunopre- cipitation using antibodies against GTF3C1 and mTOR. B, C, after SIRT7 siRNA treatment for 2 days, GTF3C1 and mTOR were immu- nopurified and analyzed via Western blotting using anti-acetyl-Lys antibody. D, HeLa cells were treated with SIRT7 siRNA for 2 days. The nucleus and nucleolus were enriched and analyzed via Western blot- ting using antibody specific to acetyl-lysine (Acetyl-K). E, SIRT7 knockdown affects mTOR pathway. Phosphorylated S6 (top panel) and LC3B protein (bottom panel) abundances were monitored via Western blotting in cells treated with siRNA. Numbers represent S6-P/S6 total (top panel) and LC3B/tubulin (bottom panel) ratios. F, STRING network of protein–protein interactions between RNA Pol III subunits and TFIIIC2 complex members. Colored nodes, reflecting relative abundance (NSAF, %), represent proteins co-isolated with SIRT7-EGFP from purified nuclei. “White nodes” added by STRING were colored gray, depicting the connectivity between RNA Pol III and

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Biomarker Discovery, Western Blot, Knockdown, Protein-Protein interactions, Isolation, Purification

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 6. Model for potential roles of SIRT7 in ribosome biogen- esis. Role of SIRT7 in Pol I, Pol III, and mTOR. SIRT7 majorly localizes within nucleoli and forms multiple complexes with ribosomes, mTOR, and TFIIIC2. Both mTOR and SIRT7 can interact with TFIIIC2 to regulate Pol III transcription. The SIRT7–mTOR complex may regulate an autophagy pathway that affects cell growth and proliferation.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: