Fig 2: Effect of ASCC2, ASCC3 or HBS1L knockdowns on general translation and PF8503-induced stalling.(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, m7G cap with m7G 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.

Fig 3: Effects of double knockdowns of ASCC3 and genes involved in translation quality control on PF8503 toxicity.(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 Fig 2A. 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.

Fig 4: Roles of ASCC2 and ASCC3 in PF8503-dependent toxicty.(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 log2(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.

Fig 5: 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 Figures S1 and S5.