Fig 2: ZNF598 and GIGYF2 mediate alternate responses to ribosome collisions.(A) Total cell lysates from WT and ∆GIGYF2 cells treated with nothing or with collision-inducing low-dose emetine (1.2 µM or 1.8 µM) were analysed by western blotting to monitor ubiquitination of eS10, the primary target of ZNF598. Note that due to its low abundance, the strip of the blot containing ubiquitinated eS10 (Ub-eS10) was detected with higher concentration of primary antibody, but is otherwise from the same membrane and gel used for the other antigens. (B) WT and ∆EDF1 cells were treated with nothing, collision-inducing low dose of emetine (1.8 µM) or stall inducing high dose of emetine (360 µM). The lysates were analysed by western blotting to assess ubiquitination of eS10 as in panel A. (C) WT and ∆GIGYF2 cells were treated with indicated siRNAs for 72 hr. Where indicated, cells were treated with 1.8 µM emetine for 15 min just prior to lysis. Shown are total cell lysates analysed by western blotting to asses ubiquitination of eS10. Note that in both cell types, eS10 ubiquitination is completely abolished when ZNF598 is knocked down. (D) Flp-In T-REx 293 with stably integrated (KAAA)21 reporter were treated with the indicated siRNAs for 3 days. After an additional 20 hr of doxycycline-induced expression of the fluorescent reporter, the cells were analysed by flow cytometry. Expression of GFP (top panel) and RFP (middle panel) was represented as a histogram for each condition plotted on a log scale. The RFP:GFP ratio (bottom panel) was calculated for each cell line using FlowJo software and also represented as a histogram for each condition.

Fig 3: Cell cycle phase–independent translation suppression upon CDK1i. (A) HeLa cells were treated with either solvent (DMSO) or the CDK1 inhibitor Ro3306 (10 µM) for 1–24 h. SG formation was analyzed by IF microscopy of fixed cells stained with anti-G3BP1 and quantified (mean ± SEM, n = 5). Statistical significance was determined by paired, one-tailed Student’s t test; scale bar = 25 µm. (B) HeLa FUCCI cells were treated with Ro3306 (10 µM) for 16 h, fixed, and analyzed for SG formation by IF microscopy upon staining with anti-eIF3B antibody. HeLa cells stably expressing Kusabira-Orange-Cdt1 (marker for G1 and early S phase) were used in the left panel; HeLa cells stably expressing mVenus-Geminin (marker for S, G2, and M phase) were used in the right panel. (C) Quantification of the percentage of SG-containing cells in Cdt1-positive (left) or Geminin-positive cells (right, n = 4). Statistical significance was determined by paired, two-tailed Student’s t test. (D) RPE-1 cells were serum-starved for 48 h and subsequently treated with DMSO or Ro3306 (10 µM) for 4 h. Polysome profiles were recorded; the percentage of polysomal ribosomes is represented in the inset (mean ± SEM, n = 3). (E) HeLa cells were arrested at the G1/S boundary by a TT block and, without release from the block, treated with either solvent (DMSO) or Ro3306 (10 µM) for 4 h. Polysome profiles were analyzed as in D (mean ± SEM, n = 3). (F) HeLa cells were synchronized as in E and released from TT block for 2, 6, 13, or 15 h. Subsequently, cells were treated with DMSO or Ro3306 (10 µM) for 2 h, and polysome profiles were analyzed as in D (mean ± SEM, n = 2). In D and E, statistical significance was determined by one-tailed paired Student’s t test, and in F by one-tailed Welch’s t test. (G) Expression of cyclins and the phosphorylation status of histone H3 (S10) from cells analyzed in F were assessed by Western blot analysis; eIF3A and tubulin levels serve as loading controls. (H) Cell cycle profiles from cells in F were analyzed by FACS using PI staining.

Fig 4: LARP1-dependent suppression of 5'TOP mRNA translation upon CDK1i. (A) For Ribo-Seq analysis, RPE1 cells were serum-starved for 48 h followed by a 4-h treatment with DMSO or Ro3306 (10 µM). Equal amounts of a yeast lysate were spiked into the DMSO- and Ro3306-treated samples. RDs (no. ribosome footprints/no. ORF-spanning reads in input RNA) were calculated after normalization to the yeast spike-in footprints from n = 3 biological repeat experiments. (B) Based on the Ribo-Seq analysis in A, the average RD was calculated after normalization (norm.) to the yeast spike-in. Statistical significance was determined by one-tailed ratio paired t test. (C) Based on the Ribo-Seq analysis in A, the fold change in RD (? RD) was calculated for IRES-containing mRNAs, 5'TOP mRNAs, and all other mRNAs. (D) Polysome association of 5'TOP (RPLP0 and PABPC4) and ORF size-matched non-TOP (EIF2S1 and NCL) mRNAs was analyzed by polysome fractionation and subsequent qPCR analysis from DMSO- or Ro3306-treated (10 µM, 4 h) HeLa cells. (E) Western blot analysis of LARP1 expression in HEK293T WT cells, HEK293T LARP1-/- cells, and HEK293T LARP1-/- + LARP1 cells expressing LARP1-Flag-SBP. RPS10 serves as loading control. (F) Polysome association of 5'TOP (RPS6 and RPS7) and ORF size-matched non-TOP (EIF2S1, CDKN1A) mRNAs was analyzed by polysome fractionation and subsequent qPCR analysis from DMSO- or Ro3306-treated (10 µM, 4 h) HEK293T WT, LARP1-/-, or LARP1-/- + LARP1 cells.

Fig 5: The shaft of the growing axon is scarcely populated by ribosomes.(A) Examples of ribosomes observed in cryo-tomograms of axon shafts from ALI-COs, of other cellular processes from ALI-COs, and of HeLa cells. The bottom panel shows 0.05 µm3 cryo-ET volumes, corresponding to the area shown in the upper panel. Positions of all ribosome-like particles observed in that volume are shown as orange spheres. (B) Comparison of the numbers of ribosome-like particles, normalized to the tomographic volume, observed in axon shafts, other processes and HeLa cells. Individual data points represent individual cryo-tomograms (30, 4, and 5 tomograms, respectively). Mann-Whitney tests were employed for statistical analysis: p < 0.0001 (****); p < 0.05(*). (C) Immunofluorescence images of dissociated neurons from organoids reveal low signal for the ribosomal 60 S component RPL8 in axon processes (identified by SMI312+/MAP2- labeling) in comparison to dendrites (identified by SMI312-/MAP2+ labeling). The yellow box outlines the area magnified in the right panel. The top image of the right panel shows the immunofluorescence signal for the ribosomal subunit RPL8. The bottom image shows the SMI312/MAP2/RPL8 composite. The white dashed line depicts the outline of axons and dendrites and was traced based on the MAP2 and SMI312 signal. The image shown is representative of the data used for quantifications shown in D. (D) Quantification of immunofluorescence images of ALI-CO derived dissociated neurons labeled for five distinct ribosomal proteins. The bars report the mean pixel grey value along axons and dendrites (mean ± SD). Each data point represents a different axon or dendrite. With the exception of quantifications done on the ribosomal protein S6, axons were identified as SMI312+/MAP2- neuronal processes, while dendrites were identified as SMI312-/MAP2+ neuronal processes. Due to antibody incompatibility, in the case of S6, dendrites were identified as MAP2+ processes while axons were identified as GFP+/MAP2- processes. The data pertains to one biological replicate. Mann-Whitney tests were employed for statistical analysis: RPL8, N = 10, p < 0.0001 (****); RPS10, N = 12, p < 0.0001 (****); RPS16, N = 12, p < 0.0001 (****); RPS26, N = 12, p < 0.0001 (****); S6, N = 12, p = 0.0001 (***). Scale bars: 50 nm in A., 20 µm and 2 µm in C.