Fig 1: Vaccinia virus replication is attenuated in cells unable to mount an RQC response. (A) ZNF598 KO or parental HCT116 or 293Flp-IN cells were infected with vaccinia virus (10,000 PFU/well, MOI ∼0.04). Cells were collected 24 h post infection, and viral titers were determined using a plaque assay. Data are presented as the mean±s.e.m. of three experiments, with individual points shown. *P<0.05 (one-way ANOVA with adjustment for multiple comparisons using Tukey's method). (B) HCT116 cells containing mutant ribosomal proteins eS10 (K138R, K139R; eS10-KI) or uS10 (K4R, K8R; uS10-KI), or parental HCT116 cells were infected with vaccinia virus (10,000 PFU/well, MOI ∼0.04). Cells were collected 24 h post infection, and viral titers were determined using a plaque assay. Three individual clones (cl1, cl2 and cl3) of uS10-KI cells were tested. Data are presented as the mean±s.e.m. of three experiments, with individual points shown. *P<0.05 (one-way ANOVA with adjustment for multiple comparisons using Tukey's method). (C) Parental or ZNF598 KO 293Flp-IN cells were mock infected (C) or infected with vaccinia virus (VacV; MOI of 5), and cells were collected at the indicated time points (hpi, hours post infection) then analyzed using SDS–PAGE and immunoblotted (IB) with antibodies against the indicated proteins (l, long exposure; s, short exposure; Ub, ubiquitylated protein). (D,E) The indicated cell lines (Par, parental) were either untreated (open circle) or treated (filled circle) with 1000 U of IFN-β for 16 h (D) or treated with the indicated concentration of IFN-β (100 U, 1000 U and 5000 U) for 16 h (E), and cells were harvested and analyzed by SDS–PAGE and immunoblotting. In C–E, size markers indicate molecular masses in kDa. Blots shown are representative of n=2 experiments.
Fig 2: USP21 and OTUD3 antagonize ZNF598-mediated RRub events.(A,B) Parental HCT116 cells and ZNF598 knock-out (KO) cells were transfected with USP21 (A) or OTUD3 (B) expression plasmids and the poly(A)-stall reporter (black circles) or the poly(A)-stall reporter alone (open circles). Fluorescence intensities were measured by flow cytometry and the relative ChFP:GFP ratio is depicted. Error bars denote SEM for triplicate transfections. ***p<0.0001, **p<0.001, *p<0.05, using Student’s t-test comparing Dubs to control transfection. (C,D) The ChFP:GFP ratio from parental HCT116 cells or point mutant knock-in (KI) eS10 or uS10 cell lines transfected with the poly(A)-stall reporter alone (open circles) or with expression plasmids for wild type (black circles) or inactive mutant (gray circles) USP21 (C) or OTUD3 (D) relative to control transfections in the indicated cell lines. Error bars denote SEM for triplicate transfections. *p<0.0001 using Student’s t-test comparing wild type Dub transfections to control transfection in the indicated cell lines. (E) Whole-cell extracts from cells transfected as indicated in panels C and D were analyzed by SDS-PAGE and immunoblotted for the indicated antibodies. Black and gray circles denote expression of wild type or inactive versions, respectively. The ubiquitin-modified ribosomal protein is indicated by the arrow. S and L denote short and long exposures (n = 1). (F,G) The ChFP:GFP ratio from HCT116 ZNF598 knockout (KO) cells transfected with increasing amounts of plasmid DNA for either wild type ZNF598 and USP21 (F) or OTUD3 (G) and the poly(A)-stall reporter. Numbers indicate the ratio of transfected DNA for each plasmid. Error bars represent SEM of triplicate replicates. ***p<0.0001, **p<0.001, *p<0.05 using Student’s t-test comparing the different ZNF598 to Dub DNA ratios as indicated.
Fig 3: Distinct sets of RRub events are hierarchically organized.(A,B) HCT116 mutant RRub knock-in (KI) cell lines eS10-KI (K138RK139R) or uS10-KI (K4RK8R) (A,B) were treated with ANS (5 μg/ml) and MG132 (10 μM) continuously for the indicated times. Whole-cell extracts were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (C,D) HCT116 mutant uS5-KI (K54RK58R) or uS3-KI (K214R) were either untreated or pre-treated with DTT (5 mM) alone or with CHX (100 μg/ml) for 4 hr followed by DTT washout in media with or without CHX (100 μg/ml) and MG132 (10 μM). Cells were collected at the indicated times post DTT washout. Whole-cell extracts were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The ubiquitin-modified and unmodified ribosomal protein is indicated by the arrow and asterisk, respectively. S and L denote short and long exposures (n = 1).
Fig 4: RQC deficiency does not specifically impact translation of mRNAs containing a 5′ UTR poly(A) sequence. (A,B) Vaccinia virus (VacV) proteins were binned as (A) early or (B) post-replicative, and the normalized log2 heavy to light (H:L) ratios of VacV proteins from parental (par; red circles, no border) or ZNF598 KO (red circles, black border) HCT116 cells infected with VacV (MOI=5) are compared at the indicated time points (hpi, hours post infection). The mean normalized log2 H:L ratio for proteins across three replicate experiments is depicted. Black bars denote the mean of all VacV H:L ratios at a given time point with error bars representing s.e.m. (C) The difference in mean normalized log2 H:L ratios for VacV early proteins (green circles) or post-replicative proteins (blue circles) between infected ZNF598 KO and parental cells is depicted. Black bars denote the mean of all H:L ratios at a given time point with error bars representing s.e.m. All mean comparisons are not significant (one-way ANOVA). (D) The mean normalized log2 H:L (uS10-KI:parental) ratios of VacV early proteins (green circles) or post-replicative proteins (blue circles) is depicted. Black bars denote the mean of all H:L ratios at a given time point with error bars representing s.e.m. All mean comparisons are not significant (one-way ANOVA). (E) Relative luminescence intensities of firefly luciferase (Fluc) reporters containing the indicated length of poly(A) sequence (0A–20A) immediately before the start codon for firefly luciferase, compared to Renilla luciferase (Rluc) controls without poly(A) sequences, is shown for uninfected (black bars) and VacV infected (red bars) cells. Mean±s.e.m. for triplicate experiments. *P<0.05 (two-tailed, unpaired Student's t-test). (F) Relative luminescence intensities of firefly luciferase reporters containing the indicated length of poly(A) sequence, as compared to Renilla luciferase controls, is shown for transfections into the indicated cell types (HCT Par, parental line for the ZNF598 KO; HCT Par-2, parental line for the eS10-KI and uS10-KI lines). Mean±s.e.m. for triplicate experiments. *P<0.05 (two-tailed, unpaired Student's t-test).
Fig 5: RNA-seq identifies intact viral and host transcriptional response in uS10-KI cells. (A) Schematic depicting the RNA-seq experimental design and analysis. Individual RNA library preparation time points are shown as a, b and c for parental HCT116 cells, and as a1, b1 and c1 for uS10-KI HCT116 cells (hpi, hours post infection; VacV, vaccinia virus). (B) Percentage mRNA alignment to the human (black bars) or VacV (blue bars) genome in parental (Par) or uS10-KI cells (C, mock-infected control sample). (C,D) Reads aligned to the VacV genome were analyzed using DESeq2, and the mean normalized counts of VacV genes, classified as early (green), intermediate (blue) or late transcribing (red), are depicted in the indicated cell lines at (C) 4 hpi. (Table S7) or (D) 8 hpi (Table S8). Dashed lines indicate the mean of the normalized counts of the plotted genes. Named genes show a similar trend of gene expression across the genotypes, across temporal expression patterns, and are plotted in a heatmap in Fig. S2B. (E) Plot of the log2-transformed ratio (uS10-KI:parental) of protein abundance (as assayed using SILAC) versus mRNA abundance (RNA-seq mean read counts) for VacV genes at 8 hpi (Table S9). Circle color indicates mRNA expression classification (green, early; blue, intermediate; red, late). (F) Scatter plot of log2 fold change (8 hpi/mock) of mRNA expression for host genes in parental cells (x axis) versus uS10-KI cells (y axis) (n=13,759) (Tables S10, S11). Selected genes are labeled. (G) Violin plot showing the frequency distribution of log2 fold-change (FC) ratios (8 hpi:mock) of the 122 common differentially expressed host genes in parental and uS10-KI cell lines when comparing uninfected cells with those at 8 h post infection.
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