Fig 2: IRES RNA affinity analysis. (a) In all, 50 μg of biotin-tagged cyclin T1 and SETD7 5' UTRs or 300 nt RNA of GAPDH coding region was used as bait to purify IRES-binding proteins from HeLa cell lysate in a binding buffer (10 mM Tris pH 7.5, 25 mM KCl, 2 mM MgCl2, 0.02% Tween-20, 1 mM ATP, 1 μg/ml yeast tRNA, 1 μg/ml heparin, RNase inhibitor, 1 × protease inhibitor) for 60 min at 4 °C. Streptavidin-conjugated magnetic beads (Invitrogen) were added and incubated for a further 30 min. The bound complexes were washed with buffers that contained increasing salt concentration and eluted samples were applied to SDS-PAGE and stained with colloidal Coomassie blue and silver. Proteins that were specifically eluted from the cyclin T1 or SETD7IRESs, but not the GAPDH are marked (*). (b) Proteins bound to each RNA were identified firstly via LC-MS/MS mass spectrometry (Supplementary Figures S2A and B) and then via immunoblotting analysis (Supplementary Figure S2C). The table shows proteins, which were identified as binding to either or both of the cyclin T1 and SETD7 IRESs, whereby a plus indicates binding, in either the mass spectrometry data, the immunoblotting data or both. The proteins included in the table are those found to be both IRES binding and PTB associated

Fig 3: SET7-mediated methylation of OTUB1 relieves its suppressive role on ferroptosis. A, quantitative real-time PCR (qPCR) analysis of PTGS2 mRNA in OTUB1-deficient H1299 cells (OTUB1−/−) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with or without TBH (100 μM) for 7 h. B, qPCR analysis of PTGS2 mRNA in OTUB1-deficient H1299 cells (OTUB1−/−) reconstituted with OTUB1 or its methylation-site mutant (OTUB1-K122A) by lentivirus and treated with or without TBH (100 μM) for 7 h. C, cell viability of OTUB1-deficient H1299 cells (OTUB1−/−) (n = 3) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with or without TBH (150 μM or 200 μM) for 7 h and examined by CCK8 assay. D, cell viability of OTUB1-deficient H1299 cells (OTUB1−/−) (n = 3) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with DMSO (as a control) or Erastin (20 μM) for 24 h and examined by CCK8 assay. E, cell viability of OTUB1-deficient H1299 cells (OTUB1−/−) (n = 3) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with or without cystine starvation for 24 h and examined by CCK8 assay. F and G, intracellular ROS in OTUB1-deficient H1299 cells (OTUB1−/−) (n = 3) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with or without Cystine starvation for 24 h and examined by flow cytometry. Quantification of the intracellular ROS levels in (F) and representative flow cytometry histogram in (G). H and I, intracellular ROS in OTUB1-deficient H1299 cells (OTUB1−/−) (n = 3) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus and treated with DMSO (as a control) or Erastin (20 μM) for 24 h and examined by flow cytometry. Quantification of the intracellular ROS levels in (H) and representative flow cytometry histogram in (I). Two-way ANOVA analysis; Data show mean ± SD; Tukey’s multiple comparisons test; ∗Adjusted p < 0.05, ∗∗Adjusted p < 0.01, ∗∗∗Adjusted p < 0.001, ∗∗∗∗Adjusted p < 0.0001; Data from three independent experiments. CCK8, Cell Counting Kit-8; ROS, reactive oxygen species; TBH, Tert-Butyl hydroperoxide.

Fig 4: SET7 has no obvious effect on EGLN1 protein level and vice versa.A, Western blot analysis of endogenous EGLN1 expression in HEK293T cells transfected with an increasing amount of Myc-SET7 expression plasmid. B, Western blot analysis of endogenous EGLN1 expression in SET7-deficient or WT HEK293T cells (SET7−/− or SET7+/+). C, Western blot analysis of endogenous EGLN1 expression in SET7-deficient HEK293T cells (SET7−/−) transfected with indicated plasmids. D, Western blot analysis of endogenous EGLN1 expression in H1299 cells transfected with an increasing amount of Myc-SET7 expression plasmid. E, Western blot analysis of endogenous SET7 expression in H1299 cells transfected with an increasing amount of HA-EGLN1 expression plasmid. F, Western blot analysis of endogenous SET7 expression in EGLN1-deficient or WT H1299 cells (EGLN1−/− or EGLN1+/+). EGLN, egg-laying defective nine; HA, hemagglutinin.

Fig 5: SET7-mediated methylation of OTUB1 does not affect OTUB1 protein stability and vice versa. A, Western blot analysis of HA-OTUB1 expression in HEK293T cells cotransfected with an increasing amount of Myc-SET7 or the enzymatically deficient mutant (Myc-SET7-H297A) expression plasmid. B, Western blot analysis of endogenous OTUB1 expression in HEK293T cells transfected with an increasing amount of Myc-SET7 or the enzymatically deficient mutant (Myc-SET7-H297A) expression plasmid. C, Western blot analysis of endogenous OTUB1 expression in SET7-deficient or WT H1299 cells (SET7−/− or SET7+/+). D, Western blot analysis of endogenous SET7 expression in HEK293T cells transfected with Flag-OTUB1 or the methylation-mimic mutant (Flag-OTUB1-K122F) expression plasmid. E, Western blot analysis of endogenous SET7 expression in OTUB1-deficient H1299 cells (OTUB1−/−) reconstituted with OTUB1 or its methylation-mimic mutant (OTUB1-K122F) by lentivirus. F, Western blot analysis of endogenous OTUB1 expression in SET7-deficient or WT H1299 cells (SET7−/− or SET7+/+) treated with CHX (50 μg/ml) for the indicated times. G, Western blot analysis of endogenous SET7 expression in OTUB1-deficient or WT H1299 cells (OTUB1−/− or OTUB1+/+) treated with CHX (50 μg/ml) for the indicated times. CHX, cycloheximide.