Fig 2: 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 3: Members of the PTB-containing complex bind apoptotic IRES RNA in vitro and in vivo. Binding of the identified complex members to the apoptotic IRES RNA was validated by a number of methods. (a) UV-crosslinking (right panels) and filter-binding assays (left panels) using 32P-labelled cyclin T1 or SETD7 5' UTR. (ai) 32P-CTP-labelled cyclin T1 and rePTB, rePSF and rePCBP2 or (aii) 32P-CTP-labelled SETD7 RNA with rePTB, rePSF and reYBX1 were used in UV-crosslinking and filter-binding assays. Specificity of the binding was demonstrated by increasing the amount of cold competitor IRES RNA (right panel), or nonspecific control RNA (left panel). The UV-crosslinked signal was quantified relative to the central no competitor RNA lane, which was set to a 100. (aiii) Dissociation constants for each protein/RNA combination were calculated in the filter-binding assay. A constant level of the 32P-labelled probe was used over a range of molarity of each recombinant protein. S.D. is shown in square brackets, and the data are the average of three repeats. (b) Binding of endogenous PTB, PSF or NONO/p54nrb to the IRES RNA was shown using RNA-IP with antibodies against the proteins indicated, followed by RT-qPCR using primers specific for cyclin T1, SETD7 or control RNA. 15 cm confluent plates of HeLa cells were lysed in lysis buffer (20 mM HEPES pH7.2, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, RNAsin 400 U/ml, 0.5% Triton X-100 and protease inhibitors), incubated for 5 min at 4 °C and centrifuged to pellet the nuclei. The post-nuclear extract was incubated with PTB, PSF or NONO/p54nrb antibodies or IgG (control) coated protein A beads for 1 h at 4 °C. Beads were washed 4 × 30 min with buffer A (20 mM Hepes pH 7.2, 200 mM NaCl, 5 mM MgCl2, 0.5% Triton X-100, 1 mM DTT) and 2 × with buffer B (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM MgCl2 and 0.05% NP40). Beads containing protein-RNA complexes were isolated and re-suspended in 200 µl buffer B and proteinase K treated for 45 min at 55 °C. The RNA was isolated by phenol/chloroform extraction and quantified by RT-qPCR using primers specific for cyclin T1, SETD7 or control RNA. Results are shown relative to a nonspecific IgG immunoprecipitation. Western blot of the IPs using antibodies against proteins indicated is shown on the right. Significance (*P<0.05, **P<0.01 or ***P<0.005) was calculated using an unpaired two-tailed Student's t-test (n=3), error bars represent S.D.

Fig 4: SET7 interacts with EGLN1.A, coimmunoprecipitation of Myc-SET7 with HA-EGLN1. HEK293T cells were cotransfected with indicated plasmids for 24 h. Anti-Myc antibody–conjugated agarose beads were used for immunoprecipitation, and the interaction was detected by immunoblotting with the indicated antibodies. B, coimmunoprecipitation of FLAG-EGLN1 with Myc-SET7. HEK293T cells were cotransfected with indicated plasmids for 24 h. Anti-FLAG antibody–conjugated agarose beads were used for immunoprecipitation, and the interaction was detected by immunoblotting with the indicated antibodies. C, endogenous interaction between SET7 and EGLN1. Anti-EGLN1 antibody was used for immunoprecipitation, and normal rabbit IgG was used as a control. D, endogenous interaction between EGLN1 and SET7. Anti-SET7 antibody was used for immunoprecipitation, and normal rabbit IgG was used as a control. E, coimmunoprecipitation of FLAG-SET7 with endogenous EGLN1. HEK293T cells were transfected with indicated plasmids for 24 h. Anti-FLAG antibody–conjugated agarose beads were used for immunoprecipitation, and the interaction was detected by immunoblotting with the indicated antibodies. F, schematic of EGLN1 domains interacted with SET7. The interaction is indicated by (?) sign. G, coimmunoprecipitation analysis of Myc-SET7 with FLAG-EGLN1–truncated mutants. HEK293T cells were cotransfected with the indicated plasmids. Anti-FLAG antibody–conjugated agarose beads were used for immunoprecipitation, and the interaction was analyzed by immunoblotting with the indicated antibodies. FLAG-EGLN1 fragments (EGLN1-C, 130–426 aa; EGLN1-N, 1–196 aa). EGLN, egg-laying defective nine.

Fig 5: SET7 methylates EGLN1 on lysine 297.A, sequence alignment of partial EGLN1 proteins (285–307 amino acids) of human, mouse, rabbit, dog, and pig. The red box indicates a conserved consensus motif (R/K-S/T-K) methylated by SET7; the blue arrow indicates the conserved lysine (K297) methylated by SET7. The amino acid is numbered based on human EGLN1 amino acid sequence. B, Western blot analysis of EGLN1 or its mutant (K297A) methylated by WT SET7 or the enzymatic-deficiency mutant of SET7 (H297A) in HEK293T cells transfected with indicated plasmids. Anti-HA antibody–conjugated agarose beads were used for immunoprecipitation and anti-EGLN1-K297Me1 antibody was used for detecting monomethyl EGLN1. C, the methylated residue in EGLN1 identified by mass spectrometry analysis. HEK293T cells were cotransfected with HA-EGLN1 and Myc-SET7 plasmids. Cell lysate was immunoprecipitated with anti-HA antibody–conjugated agarose beads overnight. Immunoprecipitated EGLN1 proteins were subjected to 8% SDS-PAGE gel, and EGLN1 bands were excised from the gel and analyzed by mass spectrometry. D, Western blot analysis of endogenous methylated EGLN1 in SET7-deficient or WT HEK293T cells (SET7-/- or SET7+/+). Anti-EGLN1 antibody was used for immunoprecipitation, and normal rabbit IgG was used as a control. Anti-EGLN1-K297Me1 antibody was used to detect monomethyl EGLN1. E, Western blot analysis of endogenous methylated EGLN1 in SET7-deficient HEK293T cells (SET7-/-) transfected with indicated plasmids. Anti-EGLN1 antibody was used for immunoprecipitation, and anti-EGLN1-K297Me1 antibody was used to detect monomethyl EGLN1. EGLN, egg-laying defective nine; HA, hemagglutinin; IgG, immunoglobulin G.