Journal: PLoS Genetics

Article Title: Cellular response to small molecules that selectively stall protein synthesis by the ribosome

doi: 10.1371/journal.pgen.1008057

Figure Lengend Snippet: (A) Comparison of the Rho phenotype in the CRISPRi screen to relative cell viability in individual gene knockouts. K562_dCas9-KRAB cells with individual sgRNAs targeting genes of interest were competed against cells with a scrambled sgRNA, in the presence of 7.5 μM PF8503. Experiments were carried out in biological triplicate with the average log 2 (fold change) and standard deviation shown. (B) Effect of treatment with PF8503 (7.5μM) on K562-dCas-KRAB cell lines expressing two different sgRNA targeting either ASCC2 or ASCC3 expression. The apoptotic index, defined as the ratio of Caspase 3/7 levels to ATP levels, measured after 6 days of 7.5 μM PF8503 or DMSO control treatment. Experiment performed in triplicate, with the average and standard deviations shown. (C) Western blot of immunoprecipitation of ASCC3 from the cytoplasm of HEK293T cells. Input, cell lysate; FT, flow-through supernatant; Wash 1 and 4, Bead washes; Elution, Proteins extracted from beads. Gel is representative of duplicate experiments. (D) Western blots obtained after isolation of 80S ribosomes from K562-dCas9-KRAB cell lines expressing scrambled sgRNA, using a sucrose cushion at low (200 mM) or high (400 mM) potassium acetate concentration. Gels are representative of experiments carried out in duplicate.

Article Snippet: Total cell lysates were incubated with 1 μg of ASCC3 antibody (Bethyl laboratories, A304-015A) for 1 hr at 4°C on a rotator.

Techniques: Comparison, Standard Deviation, Expressing, Control, Western Blot, Immunoprecipitation, Isolation, Concentration Assay

Journal: PLoS Genetics

Article Title: Cellular response to small molecules that selectively stall protein synthesis by the ribosome

doi: 10.1371/journal.pgen.1008057

Figure Lengend Snippet: (A) Metabolic labeling of ongoing translation during 30 min treatment of K562_dCas9-KRAB cells expressing scrambled sgRNA, with PF8503 (7.5 μM), DMSO control, or cycloheximide (100 μg/mL). Shown is IRDye800 labelled L-AHA incorporated into newly synthesized proteins of a representative experiment carried out in duplicate. (B) L-AHA incorporation in newly synthesized proteins in K562_dCas9-KRAB cells expressing scrambled, ASCC2, HBS1L, or ASCC3 sgRNA during 30 min treatment with DMSO or PF8503 (7.5 μM). Ratio of L-AHA incorporation for each knock-down relative to the control cell line are normalized to total protein ratio, determined by Bradford assay. Experiments were carried out in duplicate with the mean and standard deviation shown. (C) Reporter mRNAs used for cell-based assays. ARCA, m 7 G cap with m 7 G nucleotide 3’- O -methylated. 5’-UTR HBB, 5’-untranslated region of the HBB gene. PCSK9(1–35), codons 1–35 of the PCSK9 gene. (D) Inhibition of the PCSK9(1–35) reporter mRNA in K562-dCas9-KRAB cells expressing scrambled, ASCC2, HBS1L, or ASCC3 sgRNA, after 6–8 hr treatment with DMSO or various concentrations of PF8503. Experiments were carried out in biological triplicate, with mean and standard deviations at each PF8503 concentration shown.

Article Snippet: Total cell lysates were incubated with 1 μg of ASCC3 antibody (Bethyl laboratories, A304-015A) for 1 hr at 4°C on a rotator.

Techniques: Labeling, Expressing, Control, Synthesized, Knockdown, Bradford Assay, Standard Deviation, Methylation, Inhibition, Concentration Assay

Journal: PLoS Genetics

Article Title: Cellular response to small molecules that selectively stall protein synthesis by the ribosome

doi: 10.1371/journal.pgen.1008057

Figure Lengend Snippet: (A) Lentiviral construct used to generate double knockdown cell lines. For each construct, the sgRNA targeting ASCC3 was placed after the human U6 (hU6) promoter, and a second sgRNA (scrambled or targeting ASCC2, NEMF, or HBS1L) was placed after the murine U6 (mU6) promoter. (B) PF8503 phenotype (Rho) obtained in competitive growth assays performed using double knockdown cell lines (grey), compared with the rho phenotype expected from the sum of phenotypes of individual knockdowns. Due to less efficient knockdown of ASCC3 in the dual-sgRNA context, the individual phenotype of ASCC3 knockdown was taken from the cell line expressing mU6-scrambled sgRNA-hU6-ASCC3 sgRNA pair. All other phenotypes were taken from the individual knockdowns in . Experiments were carried out in biological triplicate for ASCC3-NEMF cell line and in 6 replicates for other cell lines, with mean and standard deviation shown.

Article Snippet: Total cell lysates were incubated with 1 μg of ASCC3 antibody (Bethyl laboratories, A304-015A) for 1 hr at 4°C on a rotator.

Techniques: Construct, Knockdown, Expressing, Standard Deviation

Journal: PLoS Genetics

Article Title: Cellular response to small molecules that selectively stall protein synthesis by the ribosome

doi: 10.1371/journal.pgen.1008057

Figure Lengend Snippet: (A) Comparison of gene knockdowns that had significant effects on compound toxicity (Rho phenotype) in CRISPRi screens in the presence of homoharringtonin (HHT) or PF8503. Knockdowns with significant effects in the presence of HHT alone (HHT only, grey), PF8503 alone (PF8503 only, red), or in the presence of either compound (PF8503+HHT, blue) are shown. (B) Venn diagram of significant genes in the HHT and PF8503 CRISPRi screens. (C) Pathways enriched in the common and distinct collection of genes in the HHT and PF8503 CRISPRi screens. Gene count observed corresponds to the number of genes attributed to this pathway by STRING, as a percentage of the total number of genes attributed to a pathway (D). Phenotypes obtained in K562_dCas9-KRAB competition experiments (scrambled sgRNA as control), with cells treated with three different translation inhibitors: 20 nM HHT, 7.5 μM PF8503, or 7.5 μM PF846. Included are examples predicted to be part of translation quality control (ASCC2, ASCC3, HBS1L, NEMF), RNA transport (ALYREF) and ubiquitin-mediated proteolysis (UBR5). Experiments carried in biological triplicate, with mean and standard deviation shown.

Article Snippet: Total cell lysates were incubated with 1 μg of ASCC3 antibody (Bethyl laboratories, A304-015A) for 1 hr at 4°C on a rotator.

Techniques: Comparison, Control, Ubiquitin Proteomics, Standard Deviation

Journal: Molecular Cell

Article Title: The ASC-1 Complex Disassembles Collided Ribosomes

doi: 10.1016/j.molcel.2020.06.006

Figure Lengend Snippet: ASCC Is Required for Aborting Translation at a Ribosome Stall (A) The dual-fluorescence reporter used to study translational stalling. (B) Cells were treated with control (gray shaded) or ASCC3-targeting (red traces) siRNAs for 72 h before induction of the stably integrated reporter with doxycycline for 20 h. The RFP:GFP ratio as determined by flow cytometry is plotted as a histogram. (C) Immunoblots of whole cell lysates after treatment with the indicated siRNAs for 72 h. A non-specific product detected by the ASCC1 antibody is indicated with an asterisk in this and subsequent figures. (D) Stalling assay as in (B) after treatment with the indicated siRNAs for 72 h. See also and .

Article Snippet: The ASCC3 ORF was PCR amplified from a commercially available plasmid (Origene, Cat. No RC216672) and sub-cloned into the pcDNA-3xFLAG backbone.

Techniques: Fluorescence, Control, Stable Transfection, Flow Cytometry, Western Blot

Journal: Molecular Cell

Article Title: The ASC-1 Complex Disassembles Collided Ribosomes

doi: 10.1016/j.molcel.2020.06.006

Figure Lengend Snippet: Consequence of ASCC Mutants on Terminal Stalling of Translation (A) ΔASCC2 cells with the stably integrated K( AAA ) 21 reporter were transfected with BFP alone (gray trace) or co-transfected with wild-type (WT) ASCC2 (red trace) or CUE-domain mutant ASCC2 (blue trace). After 24 h, the reporter was induced with doxycycline for 20 h before flow cytometry analysis of the transfected cells (identified by BFP expression). Parental WT cells expressing the same reporter at the same locus were analyzed in parallel (green trace). All four sets of cells were analyzed together but separated into two plots for clarity. (B) Immunoblotting for ASCC2 from cells in (A). (C) Two independent knockout clones of ASCC3 were analyzed for the expression of ASCC subunits alongside WT and ΔZNF598 cells. (D) ΔASCC3 cells with the stably integrated K( AAA ) 21 reporter were transfected with empty vector (gray trace), WT ASCC3 (red trace), or two different mutant ASCC3 constructs (blue and green traces) and analyzed by flow cytometry. (E) Immunoblots of total cell lysates from the cells in (D). See also Figure S5 .

Article Snippet: The ASCC3 ORF was PCR amplified from a commercially available plasmid (Origene, Cat. No RC216672) and sub-cloned into the pcDNA-3xFLAG backbone.

Techniques: Stable Transfection, Transfection, Mutagenesis, Flow Cytometry, Expressing, Western Blot, Knock-Out, Clone Assay, Plasmid Preparation, Construct

Journal: Molecular Cell

Article Title: The ASC-1 Complex Disassembles Collided Ribosomes

doi: 10.1016/j.molcel.2020.06.006

Figure Lengend Snippet:

Article Snippet: The ASCC3 ORF was PCR amplified from a commercially available plasmid (Origene, Cat. No RC216672) and sub-cloned into the pcDNA-3xFLAG backbone.

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Sequencing, Mutagenesis, Software, Cloning