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).