Fig. 6: ZNF598 detects ribosome collisions during translation initiation.

a, Comparison of 5′ UTR length, CDS length and transcript abundance (by TPM in RNA-seq) in mRNAs with significantly increased start site pauses (in the first five codons of the respective CDS) in ZNF598^RING-expressing inducible hiPS cells (filtered for TPM > 1; n = 702), other well-translated mRNAs included in the pause site analysis (>0.5 footprints per codon, filtered for TPM > 1; n = 2,678) and remaining mRNAs with detectable expression in inducible hiPS cells (TPM > 1 in RNA-seq; n = 10,917). P values from a two-sided Wilcoxon test; NS, not significant (P > 0.01). Box plots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× the interquartile range. b, Representative density heatmaps of 42–68-nt footprints according to length and 5′ end position around CDS start (left) and stop (right) codons in control, ZNF598 knockdown (sgZNF598) and ZNF598^RING-expressing inducible hiPS cells (top to bottom) in one of two biological replicates. c, Polysome profiling (top) and immunoblot analysis of sucrose gradient fractions (bottom) from untreated inducible hiPS cells and after treatment with 0.05 mg L⁻¹ ANS for 15 min or a short (2.5 min) or long (2 hours) treatment with 2 µg ml⁻¹ HAR (n = 1 biological replicate from two independent experiments with similar results). d, Metagene profiles of ribosomal A sites from monosome footprints around CDS start and stop codons after treatment with 5 µM 4EGI-1 (n = 2 biological replicates). e, Global protein synthesis measurements by OPP labeling in control or ZNF598^RING-expressing inducible hiPS cells before and after treatment with 5 µM 4EGI-1. Median fluorescence intensity quantified by flow cytometry (>10,000 cells per analysis) was normalized to values from untreated inducible hiPS cells (n = 4 biological replicates; P values from an unpaired two-tailed t-test).

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