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Proteintech rabbit polyclonal rnmt
$A. Localization of <t>RNMT</t> and eIF4E in U2OS cells. Confocal micrographs of cells stained with anti-RNMT and anti-eIF4E antibodies and DAPI as a nuclear marker. Single and overlaid (Ov) channels are shown. Micrographs are single sections through the plane of the cells with 63x magnification. B. eIF4E and RNMT co-immunoprecipitated in the nuclear fractions of U2OS cells. Immunoprecipitations (IP) were carried out using U2OS nuclear lysates with rabbit anti-eIF4E (eIF4E-IP) or appropriate IgG control (rIgG-IP) and analyzed by Western blotting using antibodies as indicated. Nc indicates nuclear, Sn, supernatant. C. Schematic representation of the RNMT constructs used in this study: RNMT-FL (residues 1–476), RNMT-C (164–476) and RNMT-C Δlobe (RNMT-C with residues 416–456 replaced by a GSSG linker). D. SDS-PAGE gel of overexpressed and purified RNMT and eIF4E constructs; molecular mass markers are shown. E. Crystal structure of RNMT-C (PDB 5E8J) in complex with RAM (orange) and SAH (yellow). The “lobe” region (dark green) is labelled. Nt indicates N-terminus and Ct, C-terminus. F. The two binding sites for eIF4E are shown on the crystal structure with m7GTP (PDB 1L8B), the m7GTP cap is shown as cyan sticks, W56 and W102 are shown as red spheres and blue spheres represent the charged basic residues (157,159 and 162). Residues critical for binding effector proteins at the dorsal surface (V69, W73) are shown in purple. G. GST or GST-eIF4E pulldown experiments for the following constructs, RNMT-FL, RNMT-C and RNMT-C Δlobe. H. Top, 1H-15N HSQC spectra of 50 μM 15N-labelled eIF4E in the absence (red) and presence (green) 200 μM unlabelled RNMT-C. Bottom, per residue plot of the changes in peak intensity for backbone amides and side chain indole 15NH resonances of 15N labelled eIF4E upon addition of unlabelled RNMT-C shown in the top spectrum.$
Rabbit Polyclonal Rnmt, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Identification and characterization of the interaction between the methyl-7-guanosine cap maturation enzyme RNMT and the cap-binding protein eIF4E"

Article Title: Identification and characterization of the interaction between the methyl-7-guanosine cap maturation enzyme RNMT and the cap-binding protein eIF4E

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2022.167451

$A. Localization of RNMT and eIF4E in U2OS cells. Confocal micrographs of cells stained with anti-RNMT and anti-eIF4E antibodies and DAPI as a nuclear marker. Single and overlaid (Ov) channels are shown. Micrographs are single sections through the plane of the cells with 63x magnification. B. eIF4E and RNMT co-immunoprecipitated in the nuclear fractions of U2OS cells. Immunoprecipitations (IP) were carried out using U2OS nuclear lysates with rabbit anti-eIF4E (eIF4E-IP) or appropriate IgG control (rIgG-IP) and analyzed by Western blotting using antibodies as indicated. Nc indicates nuclear, Sn, supernatant. C. Schematic representation of the RNMT constructs used in this study: RNMT-FL (residues 1–476), RNMT-C (164–476) and RNMT-C Δlobe (RNMT-C with residues 416–456 replaced by a GSSG linker). D. SDS-PAGE gel of overexpressed and purified RNMT and eIF4E constructs; molecular mass markers are shown. E. Crystal structure of RNMT-C (PDB 5E8J) in complex with RAM (orange) and SAH (yellow). The “lobe” region (dark green) is labelled. Nt indicates N-terminus and Ct, C-terminus. F. The two binding sites for eIF4E are shown on the crystal structure with m7GTP (PDB 1L8B), the m7GTP cap is shown as cyan sticks, W56 and W102 are shown as red spheres and blue spheres represent the charged basic residues (157,159 and 162). Residues critical for binding effector proteins at the dorsal surface (V69, W73) are shown in purple. G. GST or GST-eIF4E pulldown experiments for the following constructs, RNMT-FL, RNMT-C and RNMT-C Δlobe. H. Top, 1H-15N HSQC spectra of 50 μM 15N-labelled eIF4E in the absence (red) and presence (green) 200 μM unlabelled RNMT-C. Bottom, per residue plot of the changes in peak intensity for backbone amides and side chain indole 15NH resonances of 15N labelled eIF4E upon addition of unlabelled RNMT-C shown in the top spectrum.$
Figure Legend Snippet: A. Localization of RNMT and eIF4E in U2OS cells. Confocal micrographs of cells stained with anti-RNMT and anti-eIF4E antibodies and DAPI as a nuclear marker. Single and overlaid (Ov) channels are shown. Micrographs are single sections through the plane of the cells with 63x magnification. B. eIF4E and RNMT co-immunoprecipitated in the nuclear fractions of U2OS cells. Immunoprecipitations (IP) were carried out using U2OS nuclear lysates with rabbit anti-eIF4E (eIF4E-IP) or appropriate IgG control (rIgG-IP) and analyzed by Western blotting using antibodies as indicated. Nc indicates nuclear, Sn, supernatant. C. Schematic representation of the RNMT constructs used in this study: RNMT-FL (residues 1–476), RNMT-C (164–476) and RNMT-C Δlobe (RNMT-C with residues 416–456 replaced by a GSSG linker). D. SDS-PAGE gel of overexpressed and purified RNMT and eIF4E constructs; molecular mass markers are shown. E. Crystal structure of RNMT-C (PDB 5E8J) in complex with RAM (orange) and SAH (yellow). The “lobe” region (dark green) is labelled. Nt indicates N-terminus and Ct, C-terminus. F. The two binding sites for eIF4E are shown on the crystal structure with m7GTP (PDB 1L8B), the m7GTP cap is shown as cyan sticks, W56 and W102 are shown as red spheres and blue spheres represent the charged basic residues (157,159 and 162). Residues critical for binding effector proteins at the dorsal surface (V69, W73) are shown in purple. G. GST or GST-eIF4E pulldown experiments for the following constructs, RNMT-FL, RNMT-C and RNMT-C Δlobe. H. Top, 1H-15N HSQC spectra of 50 μM 15N-labelled eIF4E in the absence (red) and presence (green) 200 μM unlabelled RNMT-C. Bottom, per residue plot of the changes in peak intensity for backbone amides and side chain indole 15NH resonances of 15N labelled eIF4E upon addition of unlabelled RNMT-C shown in the top spectrum.

Techniques Used: Staining, Marker, Immunoprecipitation, Control, Western Blot, Construct, SDS Page, Purification, Binding Assay, Residue

A. GST pulldown assay of GST-eIF4E for RNMT-C with and without incubation with m7GDP reveals m7GDP does not affect the eIF4E-RNMT-C interaction. B. Top, overlay of the 1H-15N HSQC spectra of m7GDP cap (1mM) -bound 15N eIF4E (50 μM) in the presence (blue) and absence (red) of 200 μM RNMT-C, indicating that in the presence of m7GDP-cap the eIF4E-RNMT complex is still formed. Bottom, comparison of the changes in peak intensities for 50 μM 15N eIF4E in the absence and presence of 200 μM RNMT-C (green bars) and m7GDP cap (1 mM) bound 15N eIF4E (50 μM) in the absence and presence of 200 μM RNMT-C (blue bar). Peaks were normalized to 1 for RNMT-C free complexes. C. Transferred cross saturation experiments. Intensity ratio (Ion/Ioff) of RNMT-C complexed eIF4E-m7GDP amide 1H resonances with (Ion) and without (Ioff) saturation of aliphatic protons versus the same ratio in the absence of RNMT-C. Saturation was at −0.5ppm (1000 Hz bandwidth). The mean change is represented by the bold centre line, the top and bottom lines signify the mean value ± 0.66 standard deviation. D,E Location of residues identified from TCS experiments are mapped onto the surface of eIF4E (PDB, 2GPQ) and identify residues at the dorsal surface to be important in the RNMT interaction. F. 1H-13C HSQC spectrum of ILV-labelled eIF4E (50 μM) in the absence (red) and presence of RNMT-C (3.8 fold molar excess), yellow. G,H. Summary of the changes in intensity of ILV-labelled eIF4E methyl peaks induced by RNMT-C is mapped onto the structure of eIF4E, larger spheres represent larger broadening affects (see methods for details).

Figure Legend Snippet: A. GST pulldown assay of GST-eIF4E for RNMT-C with and without incubation with m7GDP reveals m7GDP does not affect the eIF4E-RNMT-C interaction. B. Top, overlay of the 1H-15N HSQC spectra of m7GDP cap (1mM) -bound 15N eIF4E (50 μM) in the presence (blue) and absence (red) of 200 μM RNMT-C, indicating that in the presence of m7GDP-cap the eIF4E-RNMT complex is still formed. Bottom, comparison of the changes in peak intensities for 50 μM 15N eIF4E in the absence and presence of 200 μM RNMT-C (green bars) and m7GDP cap (1 mM) bound 15N eIF4E (50 μM) in the absence and presence of 200 μM RNMT-C (blue bar). Peaks were normalized to 1 for RNMT-C free complexes. C. Transferred cross saturation experiments. Intensity ratio (Ion/Ioff) of RNMT-C complexed eIF4E-m7GDP amide 1H resonances with (Ion) and without (Ioff) saturation of aliphatic protons versus the same ratio in the absence of RNMT-C. Saturation was at −0.5ppm (1000 Hz bandwidth). The mean change is represented by the bold centre line, the top and bottom lines signify the mean value ± 0.66 standard deviation. D,E Location of residues identified from TCS experiments are mapped onto the surface of eIF4E (PDB, 2GPQ) and identify residues at the dorsal surface to be important in the RNMT interaction. F. 1H-13C HSQC spectrum of ILV-labelled eIF4E (50 μM) in the absence (red) and presence of RNMT-C (3.8 fold molar excess), yellow. G,H. Summary of the changes in intensity of ILV-labelled eIF4E methyl peaks induced by RNMT-C is mapped onto the structure of eIF4E, larger spheres represent larger broadening affects (see methods for details).

Techniques Used: GST Pulldown Assay, Incubation, Comparison, Standard Deviation

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). 50μM 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 (Fig 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.

Figure Legend Snippet: 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). 50μM 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 (Fig 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.

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

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 Fig. 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.

Figure Legend Snippet: 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 Fig. 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.

Techniques Used: Western Blot, Binding Assay

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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: A. Localization of RNMT and eIF4E in U2OS cells. Confocal micrographs of cells stained with anti-RNMT and anti-eIF4E antibodies and DAPI as a nuclear marker. Single and overlaid (Ov) channels are shown. Micrographs are single sections through the plane of the cells with 63x magnification. B. eIF4E and RNMT co-immunoprecipitated in the nuclear fractions of U2OS cells. Immunoprecipitations (IP) were carried out using U2OS nuclear lysates with rabbit anti-eIF4E (eIF4E-IP) or appropriate IgG control (rIgG-IP) and analyzed by Western blotting using antibodies as indicated. Nc indicates nuclear, Sn, supernatant. C. Schematic representation of the RNMT constructs used in this study: RNMT-FL (residues 1–476), RNMT-C (164–476) and RNMT-C Δlobe (RNMT-C with residues 416–456 replaced by a GSSG linker). D. SDS-PAGE gel of overexpressed and purified RNMT and eIF4E constructs; molecular mass markers are shown. E. Crystal structure of RNMT-C (PDB 5E8J) in complex with RAM (orange) and SAH (yellow). The “lobe” region (dark green) is labelled. Nt indicates N-terminus and Ct, C-terminus. F. The two binding sites for eIF4E are shown on the crystal structure with m7GTP (PDB 1L8B), the m7GTP cap is shown as cyan sticks, W56 and W102 are shown as red spheres and blue spheres represent the charged basic residues (157,159 and 162). Residues critical for binding effector proteins at the dorsal surface (V69, W73) are shown in purple. G. GST or GST-eIF4E pulldown experiments for the following constructs, RNMT-FL, RNMT-C and RNMT-C Δlobe. H. Top, 1H-15N HSQC spectra of 50 μM 15N-labelled eIF4E in the absence (red) and presence (green) 200 μM unlabelled RNMT-C. Bottom, per residue plot of the changes in peak intensity for backbone amides and side chain indole 15NH resonances of 15N labelled eIF4E upon addition of unlabelled RNMT-C shown in the top spectrum.

Article Snippet: Antibodies used in Western Blots Primary antibodies mouse RNMT (RNMT 3H3–1D12, Santa Cruz Biotechnology, Catalog No. sc-517112) 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: Staining, Marker, Immunoprecipitation, Control, Western Blot, Construct, SDS Page, Purification, Binding Assay, Residue

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: A. GST pulldown assay of GST-eIF4E for RNMT-C with and without incubation with m7GDP reveals m7GDP does not affect the eIF4E-RNMT-C interaction. B. Top, overlay of the 1H-15N HSQC spectra of m7GDP cap (1mM) -bound 15N eIF4E (50 μM) in the presence (blue) and absence (red) of 200 μM RNMT-C, indicating that in the presence of m7GDP-cap the eIF4E-RNMT complex is still formed. Bottom, comparison of the changes in peak intensities for 50 μM 15N eIF4E in the absence and presence of 200 μM RNMT-C (green bars) and m7GDP cap (1 mM) bound 15N eIF4E (50 μM) in the absence and presence of 200 μM RNMT-C (blue bar). Peaks were normalized to 1 for RNMT-C free complexes. C. Transferred cross saturation experiments. Intensity ratio (Ion/Ioff) of RNMT-C complexed eIF4E-m7GDP amide 1H resonances with (Ion) and without (Ioff) saturation of aliphatic protons versus the same ratio in the absence of RNMT-C. Saturation was at −0.5ppm (1000 Hz bandwidth). The mean change is represented by the bold centre line, the top and bottom lines signify the mean value ± 0.66 standard deviation. D,E Location of residues identified from TCS experiments are mapped onto the surface of eIF4E (PDB, 2GPQ) and identify residues at the dorsal surface to be important in the RNMT interaction. F. 1H-13C HSQC spectrum of ILV-labelled eIF4E (50 μM) in the absence (red) and presence of RNMT-C (3.8 fold molar excess), yellow. G,H. Summary of the changes in intensity of ILV-labelled eIF4E methyl peaks induced by RNMT-C is mapped onto the structure of eIF4E, larger spheres represent larger broadening affects (see methods for details).

Techniques: GST Pulldown Assay, Incubation, Comparison, Standard Deviation

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: 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). 50μM 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 (Fig 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.

Techniques: Column Chromatography, Western Blot, Incubation, Methylation, Binding Assay, 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: 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 Fig. 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.

Techniques: Western Blot, Binding Assay