Journal: Journal of molecular biology

Article Title: Identification and Characterization of the Interaction Between the Methyl-7-Guanosine Cap Maturation Enzyme RNMT and the Cap-Binding Protein eIF4E.

doi: 10.1016/j.jmb.2022.167451

Figure Lengend Snippet: Figure 3. Identification of eIF4E-RNMT complex binding sites and structural models. (A) m7GTP cap column chromatography for eIF4E in the presence or absence of RNMT-C. m7GTP cap-bound proteins were analysed using Western blotting. m7GTP-bound eIF4E binds to RNMT-C (lane 5, eIF4E protein was first bound to the m7GTP column, and subsequently incubated with RNMT-C); eIF4E-RNMT-C complex binds to m7GTP cap beads (labelled “complex” in lane 6, eIF4E was first incubated with RNMT-C to form a complex, and then incubated with m7GTP beads). RNMT-C does not bind directly to m7GTP beads (lane 4). 50 lM m7GTP elutes eIF4E from the m7GTP beads (lane 7) while non-methylated GTP did not affect eIF4E binding to m7GTP beads (lane 8), confirming specificity. Two percent input was used for eIF4E and RNMT-C. (B) GST-eIF4E pulldown assays for RNMT-C or RNMT-C/RAM complexes show that RNMT-C cannot bind eIF4E in the presence of RAM. (C) Close up view of binding site of RAM for RNMT-C (PDB 5E8J) used to guide RNMT mutagenesis experiments. Only interactions with the first two helices of RAM were considered (the RNMT-C lobe binding site is not important for eIF4E association (Figure 1(G). RNMT-C residues mutated are shown as sticks (single point mutants leading to a reduction in eIF4E binding are coloured red). (D) GST pulldown assay between GST-eIF4E and wild type and mutant RNMT-C proteins. (E) Lowest energy model of the complex of eIF4E with RNMT-C generated from Haddock. The catalytic site of RNMT (marked by SAH) and the cap-binding site of eIF4E are represented by arrows and far removed from the complex interface. (F, G) Superposition of the Haddock generated model of eIF4E and RNMT-C with (F) RNMT-RAM coordinates (PDB ID 5E8J) and (G) the eIF4E-BP1 (PDB ID 3U7X) coordinates.

Article Snippet: Antibodies used in Western blots Primary antibodies mouse RNMT (RNMT 3H31D12, Santa Cruz Biotechnology, Catalog No. sc517112) and mouse eIF4E (BD Biosciences, 610269), rabbit polyclonal RNMT (ProteinTech, 13743-1-AP), rabbit monoclonal 4E-BP1 (53H11, Cell Signaling Technology, #9644), rabbit polyclonal 4E-BP2, Cell Signaling Technology #2845, rabbit polyclonal RAM (FAM103A1 Polyclonal antibody, ProteinTech, 19422-1-AP) and GST Goat Polyclonal (Cytiva; catalog no. 45- 001-369).

Techniques: Binding Assay, Column Chromatography, Western Blot, Incubation, Methylation, Mutagenesis, GST Pulldown Assay, Generated

Journal: Journal of molecular biology

Article Title: Identification and Characterization of the Interaction Between the Methyl-7-Guanosine Cap Maturation Enzyme RNMT and the Cap-Binding Protein eIF4E.

doi: 10.1016/j.jmb.2022.167451

Figure Lengend Snippet: Figure 4. eIF4E-RNMT, eIF4E translation, and export complexes are mutually exclusive. (A, B) GST or GST- eIF4E pulldowns for RNMT-C in the presence or absence of 4E-BP1, 4E-BP2, 4Gp (a peptide of eIF4G, see text) (A) or LRPPRC (B). Western blots are probed as indicated. Controls for GST binding are given in Supplementary Figure S6. (C) Schematic model of summarizing the complexes explored in A and B. RNMT, LRPPRC, eIF4G and the 4EBPs all bind overlapping surfaces on eIF4E and thus form mutually exclusive complexes. The multiple arrows between the RNMT and RNA export complexes indicate that there could be other complexes between the capping and export ones depicted. Not all eIF4E complexes known are shown here for simplicity.

Article Snippet: Antibodies used in Western blots Primary antibodies mouse RNMT (RNMT 3H31D12, Santa Cruz Biotechnology, Catalog No. sc517112) and mouse eIF4E (BD Biosciences, 610269), rabbit polyclonal RNMT (ProteinTech, 13743-1-AP), rabbit monoclonal 4E-BP1 (53H11, Cell Signaling Technology, #9644), rabbit polyclonal 4E-BP2, Cell Signaling Technology #2845, rabbit polyclonal RAM (FAM103A1 Polyclonal antibody, ProteinTech, 19422-1-AP) and GST Goat Polyclonal (Cytiva; catalog no. 45- 001-369).

Techniques: Western Blot, Binding Assay