Fig 2: eIF2α phosphorylation and noncanonical start site translation increase with altered ribosomal components upon FXR1 overexpression.(A) RP levels by qPCR normalized to actin mRNA in FXR1 KD and in FXR1-OE compared to control cells (table S3C). Western blots of (B) RPLP0, (C) RPLP2, and (D) RPL19 and RPL29 in FXR1-OE and control cells. (E) Y10B immunoprecipitation from in vivo–crosslinked cytoplasmic extracts, followed by DEAE fractionation to analyze ribosome complex migration in salt fractions in G0 and AraC-treated cells compared to S+ cells by qPCR for 18S and 28S rRNAs (representing 80S ribosomes) comigration. (F) DEAE fractionation and qPCR of rRNAs of Y10B-immunopurified ribosome complexes (18S, 28S rRNAs) in the 1 M fraction normalized to 500 mM fraction, from G0, and AraC-treated cells, versus S+ cells; FXR1-OE cells versus control cells. (G) Western blots of phospho-PKR in FXR1-OE, AraC-treated versus control cells. (H) snoRA2A and snoRD46 overexpression, followed by Western blot for phospho-eIF2α. Western blot of (I) phospho-GCN2 and (J) phospho-eIF2α in FXR1-OE versus control cells. (K) GCN2 immunoprecipitation, followed by rRNA qPCR; Western blot of RPLP0 and GCN2 (In = 10% input), in FXR1-OE versus control cells. (L) Western blot of phospho-eIF2α in FXR1 KD versus control cells. (M) Western blot of phospho-GCN2 and phospho-eIF2α in FXR1-OE cells transfected with control or RPLP0 shRNAs. (N) Translation ratios of GUG start site luciferase over AUG reporter normalized to cotransfection Renilla. G0, AraC-treated compared to S+ cells. (O) FXR1-OE compared to control cells. Data are average of three replicates ± SEM. See also fig. S3 and table S3.

Fig 3: RNA-independent activation of PKR triggers clustering and induces ISR. (A) Quantitative real-time PCR analysis of GADD34 and CHOP levels after forced-dimerization of FKBP-PKR (mean and SEM, N = 5 experiments). (B) Representative immunofluorescence images showing colocalization of FLAG-tagged FKBP-PKR and Edc3. Image crop on right: close-up. Scale bar: 10 µm. (C) Quantification of the number of catalytically dead FKBP-PKR clusters 10 min after dimerizer addition (N = 3 experiments, n > 1,000; **, P < 0.01; ****, P < 0.0001, unpaired Student’s t test, nonparametric).

Fig 4: mRuby-PKR forms clusters in response to synthetic and natural inputs. (A) Representative time-lapse micrographs showing discrete mRuby-PKR clusters in H4 cells transfected with poly I:C. mRuby-PKR in clusters comprises 5.06% ± 0.2 of the total amount of protein (see Materials and Methods for details). Scale bar: 10 µm. (B) Violin plots showing the maximum number of mRuby-PKR clusters per cell in H4 cells transfected with poly I:C (N = 5 experiments, n > 200 cells). (C) Representative live-imaging time-lapse micrographs showing merging and segregation of mRuby-PKR clusters. Scale bar: 2 µm. (D) Representative immunofluorescence image showing that phosphorylated mRuby-PKR is enriched in clusters. Scale bar: 10 µm. (E) Representative immunofluorescence images of wild-type H4 cells treated with poly I:C and immunostained for endogenous PKR. (F) Representative micrograph showing formation of mRuby-PKR clusters in measles-infected (strain MVvac-CKO-GFP) H4 cells. The inset corresponds with the outlined cell in the high-magnification image. Note that the uninfected cell in the top left corner shows no mRuby-PKR clusters. Scale bar: 10 µm. (G) Quantification of de novo mRuby-PKR clustering frequency (red bars) and MVvac-CKO-GFP replication (GFP MFI and 95% confidence interval bands) as function of time. Red trace, nonlinear curve fit of the mRuby-PKR clustering frequency data. The percentage of infected cells that formed mRuby-PKR clusters was, on average, 45 ± 5% (N = 3 experiments, n > 400 cells). (H) Quantification of normalized mRuby-PKR cluster fluorescence intensity in 1,6-hexandiol–treated H4 cells. T0 corresponds to 60 min of poly I:C treatment. The data were binned and are shown as the mean and 95% confidence interval bands (n = 60 cells). The micrographs show representative images of mRuby-PKR cluster dissolution by 1,6-hexandiol treatment. Scale bar: 10 µm. (I) FRAP analysis showing the recovery of normalized fluorescence intensity of mRuby-PKR clusters. The data are shown as in H (N = 3 experiments, n = 30 cells). The micrographs show representative images of a single mRuby-PKR cluster photobleached with a 561-nm laser beam.

Fig 5: Generation of a stable cell line expressing fluorescently tagged PKR. (A) Schematic representation of the expression construct encoding mRuby- and FLAG-tagged PKR of human origin. The mRuby fluorescent protein (236 aa) was inserted between residues 221 and 222 of human PKR. (B) Representative micrograph showing that mRuby-PKR is a cytosolic soluble protein. Scale bar: 10 µm. (C) Western blot showing the level of expression of mRuby-PKR compared with that of endogenous PKR. The relative protein amount determined by densitometry is shown below the blots. Endogenous PKR was depleted using CRISPRi. GAPDH, loading control. (D) Western blot analysis showing endogenous PKR induction in wild-type H4 cells treated with IFNβ for 16 h. GAPDH, loading control. Metrics as in C. (E) Western blot analysis comparing the activity and kinetics of endogenous PKR and mRuby-PKR in H4 cells treated with poly I:C. (F) Violin plots showing the diameter of mRuby-PKR clusters in H4 cells stably expressing mRuby-PKR and treated with poly I:C. (G) Quantification of the number of cells with endogenous PKR and mRuby-PKR clusters upon 90 min of poly I:C treatment (N = 3 experiments, n > 800; unpaired Student’s t test, nonparametric). (H) Representative time-lapse micrographs showing the formation of mRuby-PKR clusters (yellow arrowheads) in dividing H4 mRuby-PKR cells synchronized with thymidine. Scale bar: 10 µm. (I) Quantification of the half-life of mRuby-PKR in clusters after photobleaching. (J) Quantification of the mRuby-PKR immobile fraction in clusters after photobleaching. For H and I, N = 3 experiments, n = 30. Source data are available for this figure: SourceData FS1.