Journal: eLife

Article Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

doi: 10.7554/eLife.90762

Figure Lengend Snippet: ( a ) Schematic of the isNS1 batch immunoaffinity purification protocol and the downstream analyses used in this article. Infected cell supernatant from Vero cells (either WT or T164S EDEN2) was harvested at 72 hpi, clarified, filtered, supplemented with a protease inhibitor cocktail and 0.05% sodium azide, and finally concentrated using a 100 kDa MWCO Vivaflow cassette attached to a peristaltic pump. isNS1wt or isNS1ts was then batch immunoaffinity purified using the 56.2 anti-NS1 antibody immobilized on the AminoLink resin. The resin was then loaded into a column, washed with at least 10 CV of PBS (pH 7.4), eluted with 0.1 M glycine (pH 2.7), and immediately neutralized with 1 M Tris-HCl (pH 9.0). The eluted protein was then dialyzed against PBS and concentrated using a 100 kDa MWCO Amicon ultracentrifugal unit and stored at –80°C before use. The protein purity was determined via Coomassie blue after separation on a reducing SDS-PAGE. The protein bands observed on the gel were then validated in a western blot against NS1 and ApoA1. Protein quality was also determined via NS1 western blot following separation on a Native-PAGE. Excised bands corresponding to 250 kDa on the Native gel, and 50 kDa and 25 kDa bands on the denatured gel as well as the elute in solution were also subjected to protein identification via liquid chromatography mass spectrometry (LC-MS). Purified isNS1wt and isNS1ts were separately complexed with Fab56.2 and Ab56.2 and analyzed through an analytical size exclusion chromatography to ensure formation of stable complexes for imaging via electron microscopy. Purified isNS1wt and isNS1ts were also crosslinked with disuccinimidyl sulfoxide (DSSO) to determine interaction sites between isNS1 and ApoA1 via LC-MS. ( b ) Enrichment of NS1 during the immunoaffinity purification process. isNS1wt purification is used as a representative for the isNS1 purification process. The proportion of NS1 over total protein was measured by taking a percentage of the total amount of NS1 measured (using NS1 ELISA) out of the total protein measured (using Bradford assay) in the crude supernatant before concentrating, crude supernatant after concentrating, immunoaffinity PBS wash and finally in the immunoaffinity elute after buffer exchange against PBS and further concentrating. There is an approximately 455-fold enrichment of NS1 from the concentrated crude supernatant to the immunoaffinity elute. ( c ) Representative negative stain electron micrograph of isNS1 image on a 120 kV FEI Tecnai T12 equipped with an Eagle 4 mega pixel CCD camera. The corresponding 2D classes from the particles picked are shown on the right, red arrows highlighting the NS1 dimer protruding out of the spherical density.

Article Snippet: Vero cells (green African monkey kidney epithelial cells, ATCC) were cultured in DMEM containing 4.5 g/L glucose (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (P/S) at 37°C in 5% CO 2 .

Techniques: Immunoaffinity Purification, Infection, Protease Inhibitor, Purification, SDS Page, Western Blot, Clear Native PAGE, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Size-exclusion Chromatography, Imaging, Electron Microscopy, Enzyme-linked Immunosorbent Assay, Bradford Assay, Buffer Exchange, Staining

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: Density of UG nucleotide sequences 100bp upstream and downstream of m6A modifications identified by cross-linking induced mutation sites (CIMS; A ) or cross-linking induced truncation sites (CITS; B ) in relation to random sequences (red line). Grey shading represents 95% confidence regions. ( C ) Schematic of HaloTag immunoprecipitation and dot blot procedure. ( D ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of HaloTag-labeled proteins in HEK293T cells overexpressing HaloTag, TDP43-HaloTag or YTHDF2-HaloTag from 3 biological replicates. ( E ) Diagram illustrating insertion of the HaloTag open reading frame into the endogenous TARDBP locus immediately 5’ to the TDP43 start codon, resulting in a fusion of HaloTag to the N-terminus of TDP43. ( F ) Halo-TDP43 HEK293T cells labeled live with JF646 Halo dye (red), then fixed, permeabilized, and immunostained with anti-TDP43 antibody (green) prior to imaging. DAPI (blue) marks the nucleus of each cell. Scale bar = 10µm. ( G ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of endogenous HaloTag-TDP43 or exogenous HaloTag. Additional replicates shown in Sup. Fig. 1.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Mutagenesis, Immunoprecipitation, Dot Blot, Modification, Isolation, Immunoaffinity Purification, Labeling, Imaging

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) HaloTag-TDP43 immunoprecipitation was followed by DART-seq to delineate m6A sites within TDP43 target RNAs. HaloTag-TDP43 HEK293T cells were transfected with APOBEC1-YTH or APOBEC1-YTHmut and crosslinked before immunoaffinity purification of HaloTag-labeled proteins. Immunoprecipitated RNAs were then sequenced and C-T transitions were identified in the context of DRACH motifs (red shaded box, D=A/G/T, R=A/G, H=A/C/T). Absolute counts ( B ) and relative frequency ( C ) of base pair transitions observed by RNA-seq in each condition. Shaded boxes represent transition types expected from APOBEC1 activity. ( D ) Example m6A sites identified by DART-seq in RPL10A . C-T transitions are highlighted in red, and DRACH motifs in pink. Green arrow, transcription start site; red hexagon, transcription stop site; thick blue bars, coding exons; thin blue bars, untranslated region. ( E ) Absolute count and relative distribution ( F ) of DART-seq reads in cells expressing APOBEC1-YTH and APOBEC1-YTHmut. UTR, untranslated region; CDS, coding sequence. ( G ) Scatter plot of TDP43 targets, determined by fold enrichment in precipitated RNA from HaloTag-TDP43 cells (expressing APOBEC1-YTH and APOBEC10YTHmut) compared to cells transfected with HaloTag. Red dots signify transcripts showing > 2-fold enrichment in both APOBEC1-YTH and APOBEC1-YTHmut expressing cells. TARDBP , yellow dot, identified as high confidence target. ( H ) Stacked bar graph showing percentage of m6A modified RNA in TDP43 targets (red) and non-targets (black). ( I ) Cumulative distribution of RNA methylation in TDP43 targets (red) and non-targets (black). p = 1.87×10 −55 by Kolmogorov Smirnov test. ( J ) Euler diagram depicting overlap between TDP43 targets identified in this study, and those identified by TDP43 cross linking and immunoprecipitation followed by RNA-sequencing (CLIP-seq) in HEK293T cells (Hallegger et al ., 2021) . **p=1.5×10 −117 , hypergeometric test. ( K ) Pie charts demonstrating the percentage of methylated RNA among TDP43 targets (pink) and non-targets (grey). **p<1×10 −5 chi-square test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunoprecipitation, Transfection, Immunoaffinity Purification, Labeling, RNA Sequencing, Activity Assay, Expressing, Sequencing, Modification, Methylation

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) TARDBP gene map, illustrating TDP43 binding region (TBR), the location of the DRACH motif (pink square), and the C-T transition (red box) identified by DART-seq within this domain, representing an m6A site. ( B ) Schematic of the TARDBP minigene reporter, consisting of the mCherry ORF upstream of TARDBP exon 6 and 3.4 Kb of the TARDBP 3’ UTR. The A residue adjacent to the detected C-T transition via DART-seq in the WT reporter (mCherry-TBR) was mutated to a G, precluding methylation the mutant reporter (mCherry-mTBR). Red, methylated residue; blue line, DRACH motif; dagger, C-T transition from DART-seq. ( C ) HaloTag-TDP43 was isolated by immunoaffinity purification from HaloTag-TDP43 HEK293T cells expressing mCherry-TBR or mCherry-mTBR, and reporter RNA detected in elution fractions by qRT-PCR. ( D ) Outline of TDP43 autoregulation assay. Excess TDP43 binds to the reporter, triggering reporter splicing, destabilization, and reduced mCherry fluorescence. ( E ) Primary rodent neurons were transfected with WT (mCherry-TBR) or mutant (mCherry-mTBR) reporters, together with EGFP or TDP43-EGFP. After 7d, mCherry expression was assessed by fluorescence microscopy. Scale bar= 20 µm. Normalized RFP (mCherry) intensity in primary neurons expressing WT mCherry-TBR reporter ( F ) or mutant mCherry-mTBR ( G ) reporter together with EGFP or TDP43(WT)-EGFP. Cherry-TBR+GFP n= 160, Cherry-TBR+TDP43(WT)-GFP n= 58, Cherry-mTBR+GFP n= 105, Cherry-mTBR+TDP43(WT)-GFP n= 44. Data in C plotted as mean ± SD, collected from 3 biological replicates. ns= not significant, *p< 0.05, **p< 0.01; one-way ANOVA with Tukey’s test. Data in F and G plotted as mean ± SD, color coded by biological replicate. ns = not significant, *p < 0.05; Welch’s t-test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Binding Assay, Residue, Methylation, Mutagenesis, Isolation, Immunoaffinity Purification, Expressing, Quantitative RT-PCR, Fluorescence, Transfection, Microscopy

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Genome-wide analysis of RNA methylation via epitranscriptomic array. RNA was extracted from control (n= 3) and sporadic ALS (sALS) patient (n= 4) spinal cord samples, prior to m6A RNA immunoprecipitation. The resulting samples were separated into methylated and non-methylated RNA, then labeled with distinct fluorescent dyes (red and green stars) prior to hybridization, allowing relative quantification of methylation at each annotated locus. ( B ) Principal component analysis (PCA) plot comparing methylation levels of control (grey) and ALS (red) patient samples. ( C ) Hierarchical clustering of mRNA methylation profiles from control and ALS mRNA samples. ( D ) Volcano plot depicting fold change in mRNA methylation levels in ALS compared to control spinal cord. ( E ) Hierarchical clustering of lncRNA methylation profiles from control ALS lncRNA samples. ( F ) Volcano plot showing fold change in lncRNA methylation levels in ALS compared to control spinal cord. In D and F , grey horizontal vertical lines represent p= 0.05 and fold change (FC)= 2. ( G ) Euler diagram demonstrating overlap (n= 322, p= 5.09×10 −119 , hypergeometric test) among TDP43 substrates and methylated transcripts identified in HEK293T cells, in additional to hypermethylated transcripts determined via m6A array in sALS spinal cord. Comparisons were limited to the subset of transcripts expressed in both HEK293T cells and human spinal cord (nTPM>2). ( H ) Based on comparisons with the GEO transcription factor loss-of-function database via Enrichr , there was strong enrichment for TDP43-regulated genes not only among the set of 2034 transcripts hypermethylated in sALS spinal cord, but also among the 322 TDP43 targets that were also hypermethylated in sALS (A1 in G ). Combined score = (log 10 p * Z-score). ( I ) Immunohistochemical staining for m6A in control and sALS spinal cord sections. Scale bars= 50 µm. ( J ) Quantification of m6A antibody reactivity in spinal cord neurons from control (n= 110 neurons) and sALS (n= 277 neurons) sections. Plot shows mean +/- SD, color coded by patient. ****p< 0.0001 via Mann-Whitney test.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Genome Wide, Methylation, Control, RNA Immunoprecipitation, Labeling, Hybridization, Quantitative Proteomics, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Representative images of rodent primary neurons transfected with plasmids expressing Cas9-2A-EGFP and sgRNA targeting the neuronal protein NeuN or negative control (LacZ). 5d after transfection, neurons were fixed and immunostained for NeuN (red). White dashed circles indicate nucleus stained with Hoechst (blue). ( B ) NeuN antibody reactivity measured in EGFP-positive neurons expressing sgLacZ (n= 565) or sgNeuN (n= 654), ****p < 0.0001 by Mann-Whitney. ( C ) Schematic depicting m6A writers (green), erasers (red), and readers (orange) targeted by CRISPR/Cas9. ( D ) Primary neurons expressing EGFP and TDP43-mApple were assessed at regular 24h intervals by fluorescence microscopy, and their survival assessed by automated image analysis. Individual neurons are assigned unique identifiers (yellow number) and tracked until their time of death (red), indicated by cellular dissolution, blebbing, or neurite retraction. Scale bar= 20µm. ( E ) Cumulative hazard plot depicting risk of death for neurons expressing TDP43(WT) + non-targeting (NT) (red line), mApple + NT (grey line), or TDP43(WT) + Atxn2 sgRNA (purple line). †p<2.0 ×10 −16 , Hazard ratio (HR)= 3.45; ***p= 5.81 ×10 −4 , HR= 0.80). ( F ) Forest plot showing HR for TDP43-overexpressing neurons upon knockdown of m6A writers (green), erasers (dark red), and readers (orange), in comparison to nontargeting (NT) control. Dashed line indicates HR= 1, representing the survival of the reference condition, neurons expressing TDP43-mApple and NT sgRNA. Values >1 indicate increased toxicity, whereas values <1 denote relative protection. Error bars represent 95% CI. ( G ) Alkbh5 knockout significantly increases TDP43 associated toxicity. †p=3.11 ×10 −5 , HR= 1.59; ***p= 2.65×10 −11 , HR= 2.03. ( H ) Ythdf2 knockout significantly extends survival in TDP43-expressing neurons. ***p <2.0 ×10 −16 , HR= 1.69; †p= 6.2 ×10 −6 , HR= 0.71. ( I ) YTHDF2 overexpression is toxic to neurons. ***p= 3.07×10 −5 , HR= 1.30. ( J ) METTL3/14 overexpression enhances TDP43-dependent toxicity in neurons. †p = 5.53 ×10 −4 , HR= 1.32; ***p =4.16 ×10 −6 , HR= 1.31. p values in E, G-J determined via Cox proportional hazards analysis, with a minimum 3 of biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Transfection, Expressing, Negative Control, Staining, MANN-WHITNEY, CRISPR, Fluorescence, Microscopy, Dissolution, Knockdown, Comparison, Control, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Immunostaining of YTHDF2 in control and sALS patient spinal cord samples. Scale bar= 50 µm. ( B ) Quantification of YTHDF2 immunoreactivity in spinal cord neurons from control (n= 117 neurons) and sALS (n= 193 neurons) samples. Plot shows mean +/- SD, color coded by sample. ****p< 0.0001 via Mann-Whitney test. ( C ) Strategy used to create isogenic iPSCs expressing native TDP43(WT)-Dendra2 or TDP43(M337V)-Dendra2. ( D ) Representative images of untransduced (grey) and transduced (green) iNeurons expressing shRNA against YTHDF2 (shYTHDF2) and a GFP reporter. Time of death (red circles) for each cell is used to determine cumulative risk of death, plotted in ( E ) and ( F ). Scale bar= 20µm. shRNA-mediated knockdown of YTHDF2 significantly extended the survival of TDP43(M337V)-Dendra2 iNeurons ( E ; †p= 8.42×10 −12 , HR= 6.25; ***p= 4.82×10 −9 , HR=0.32; #p= 0.08, HR= 1.84) as well as mutant C9ORF72 iNeurons ( F , †p= 1.42×10 −11 , HR= 2.85; ***p= 1.42×10 −16 , HR= 0.32). ns= not significant. Values in ( E , F ) calculated by Cox proportional hazards analysis, with a minimum 3 biological replicates.

Article Snippet: Coverslips were then incubated overnight with blocking buffer + rabbit anti-TDP43 antibody (Proteintech, #10782-2-AP) at 1:500 to stain for TDP43.

Techniques: Immunostaining, Control, MANN-WHITNEY, Expressing, shRNA, Knockdown, Mutagenesis

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: