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The post-translational methylation of α-amino groups was first discovered over 30 years ago on the bacterial ribosomal proteins L16 and L331–2, but almost nothing is known about the function or enzymology of this modification. Several other bacterial and eukaryotic proteins have since been shown to be α-N-methylated3–10. However, the Ran guanine nucleotide-exchange factor, RCC1, is the only protein for which any biological function of α-N-methylation has been identified3, 11. Methylation-defective mutants of RCC1 have reduced affinity for DNA and cause mitotic defects3, 11, but further characterization of this modification has been hindered by ignorance of the responsible methyltransferase. All fungal and animal N-terminally methylated proteins contain a unique N-terminal motif, Met-(Ala/Pro/Ser)-Pro-Lys, indicating they may be targets of the same, unknown enzyme3,12. The initiating Met is cleaved, and the exposed α-amino group is mono-, di-, or trimethylated. Here we report the discovery of the first α-N-methyltransferase, which we named N-terminal RCC1 methyltransferase (NRMT). Substrate docking and mutational analysis of RCC1 defined the NRMT recognition sequence and enabled the identification of numerous new methylation targets, including SET/TAF-I/PHAPII and the retinoblastoma protein, RB. Knockdown of NRMT recapitulates the multi-spindle phenotype seen with methylation-defective RCC1 mutants3, demonstrating the importance of alpha-N-methylation for normal bipolar spindle formation and chromosome segregation.
To purify the RCC1 N-terminal methyltransferase, soluble HeLa nuclear extract was fractionated over hydroxyapatite13,14, 15 (Fig. 1a). Step-elutions were performed with increasing sodium phosphate. Fractions were assayed for activity by immunoblotting methyltransferase assays with anti-me2-SPK or by ELISA assay (Supplementary Fig. S3a). RCC1 methylation activity eluted in the 40 – 60 mM fractions (Fig. 1b). The 40 mM fraction was analyzed by mass spectrometry (MS). Among peptides for >100 genes, 2 were detected and manually confirmed for an uncharacterized methyltransferase, METTL11a/C9orf32/Ad-00316 (Gene ID: 28989). METTL11a (now renamed NRMT) encodes a 25 kDa protein in the methyltransferase 11 family, most members of which methylate metabolites or other small molecules. NRMT lacks a SET domain but possesses a Rossman-like α/β fold. According to GeneNote and Oncomine, it is ubiquitously expressed in normal tissue and robustly over-expressed in gastrointestinal cancers. It has been conserved throughout eukaryotic evolution (Supplementary Fig. S1), but next to nothing is known about the function of NRMT orthologues in any model organism.
To determine whether NRMT is the authentic RCC1 α-N-methyltransferase, we over-expressed it in HEK 293LT cells and tested nuclear extracts for RCC1 methylation activity by ELISA. Over-expression of NRMT increased α-N-methylation 3-fold as compared to a pK-YFP transfected control (Fig. 1c). Similar results were obtained using N-terminally tagged FLAG-NRMT (Fig. 1c). FLAG-NRMT immunoprecipitated from 293LT cells and eluted with FLAG peptide also methylated recombinant RCC1-His6 in vitro (Fig. 1f). The methylation was verified to be on the N-terminal Ser by Fourier transform mass spectrometry (FTMS) (Supplementary Fig. S2a). This method was used because standard approaches cannot readily distinguish between trimethylation and acetylation. Depleting NRMT in 293LT cells, using lentivirus, significantly decreased methylation of endogenous RCC1, while not affecting overall RCC1 level (Fig. 1d). Similar results were obtained by depleting NRMT in HeLa cells using short interfering RNAs (siRNAs) (Supplementary Fig. S3b). Rabbit polyclonal antibodies generated against a unique C-terminal NRMT peptide confirmed effective knockdown of NRMT levels by the lentivirus and siRNAs (Fig. 1d and Supplementary Fig. S3b). Control virus had no effect as compared to untransfected 293LT cells (Supplementary Fig. S3c). Importantly, RCC1 methylation was rescued by expression of murine NRMT-FLAG, which is not targeted by the human shRNA (Fig. 1e), confirming that off-target effects of the RNAi were not responsible for the loss of methylation. Together, these data conclusively prove that NRMT is the predominant α-N-methyltransferase for RCC1.
Rabbit polyclonal antibody (anti-me2-PPK) against a methylated peptide corresponding to mouse RCC1 also detected methylation by NRMT of RCC1 possessing a Pro2 residue (Fig. 1g). PPK-RCC1 is present in all mammalian species except humans and chimpanzees. RCC1 methylation activity was originally found in the nuclear extract of HeLa cells3 and immunofluorescence of endogenous NRMT, or imaging of a NRMT-GFP fusion protein, showed the enzyme is predominantly nuclear (Fig. 1h and Supplementary Figs. S3d, e). siRNA against NRMT abolished the nuclear staining, confirming this localization pattern (Fig. 1h). Together, these data prove that NRMT is the authentic α-N-RCC1 methyltransferase and that it can recognize variants of the consensus sequence from different species.
The crystal structure of NRMT in complex with S-adenosyl-homocysteine (SAH) was solved to 1.75 Å resolution by the Structural Genomics Consortium (PDB entry 2EX4, at http://www.pdb.org/pdb/explore/explore.do?structureId=2EX4). A large cavity opposite the SAH binding site could accommodate N-terminal peptides (Fig. 2a) and contains an arrangement of aromatic residues similar to those in chromo domains. We used ICM-PRO to model an RCC1 N-terminal peptide (Ser-Pro-Lys-Arg-Ile-Ala) in the putative active site (Fig. 2a). In this model, only the first 3 residues (Ser-Pro-Lys) interact with NRMT. The optimum conformation positions the substrate α-NH2 close to the SAH, in the correct orientation for methyl transfer, within 3.6Å of the sulfur atom. The peptide Lys4 side chain forms hydrogen bonds and electrostatic interactions with acidic residues at the lip of the active site. A stabilizing effect on Lys4 results mainly from Asp178 and Asp181 (2.9Å, and 3.4Å from the ε-NH2, respectively) plus a weaker effect of Ser183 (Fig. 2b). A similar structural motif was reported previously to coordinate lysine residues17. Other peptides, with Pro2 or Ala2, adopt the same conformation (data not shown); however, substitution of Lys4 by Gln prevented interactions with the basic residues at the lip of the active site (Fig. 2c).
To test the requirement for Lys4 in substrate binding to NRMT, we measured its affinity, by isothermal titration calorimetry, for wild-type RCC1 N-terminal peptide and a mutant peptide in which Gln replaced Lys4. Wild-type peptide showed exothermic binding to NRMT (ΔH= −9.8kcal/mol, Kd 70µM), while RCC1(Gln4) did not bind detectably (Fig. 2e). Lys4 is, therefore, an essential determinant of NRMT substrate specificity. In vitro methylation assays confirmed that only the wild-type peptide can be methylated by recombinant NRMT (Supplementary Fig. S3f). A second key interaction involves H-bonding between the Pro3 carbonyl and the Asn169 amido group (Fig. 2b). NRMT(Lys169) had no activity (Fig. 2d), while mutation of Asp168, which does not interact with the peptide substrate, had no effect on methylation (Fig. 2d). Mutating residues (Asp178, Asp181) at the lip of the active site to Ala decreased enzyme activity, which was further decreased by reverse-charge mutagenesis to Lys (Fig. 2d). Mutating Ser183 to Lys also decreased activity (Fig. 2d). Together, these data strongly support the model for substrate binding predicted from the structure and docking analysis.
We found previously that α-N-methylation occurred on RCC1 containing Ser2, Pro2 or Ala23. To extend this analysis, we mutated the second residue to each of the other 17 amino acids, using a system in which Factor X cleavage provides efficient exposure of this residue. Testing the cleaved proteins for methylation revealed the enzyme to be promiscuous. Only acidic residues, some hydrophobic residues, and Trp gave no detectable methylation (Fig. 3a). Downstream of the x-Pro-Lys motif, mutagenesis had variable effects, but did not abolish methylation by NRMT (data not shown). We conclude that the x-Pro-Lys motif is important for recognition by NRMT, but that substrate specificity is likely also controlled by the efficiency with which the initiating Met is cleaved.
Using Met-(Ala/Ser/Pro)-Pro-Lys, we searched Genbank for candidate substrates of NRMT. More than 35 annotated genes contain this N-terminal motif (Supplementary Table 1). To screen tissues for α-N-methylated proteins, we immunoblotted mouse tissue lysates with our me3-SPK and me2-PPK antibodies. In addition to RCC1, the immunoblots picked up >10 other proteins (Supplementary Fig. S4a). All tissues contained RCC1 N-methylation activity (Supplementary Fig. S4b). To validate the Genbank search, we asked if any proteins detected by the antibodies corresponded to predicted substrates. Proteins were precipitated from HeLa cell lysates using anti-me3-SPK and separated by SDS-PAGE. Besides RCC1 and antibody chains, one additional band was visible at a size also detected by immunoblotting (Fig. 3b). This band was identified by MS as SET/TAF-I/PHAP-II, a predicted substrate for NRMT (Supplementary Table 1). SET has two splice variants, α and β. Only SET α begins with the NRMT consensus. When C-terminally tagged SETα-GFP and SETβ-GFP were expressed in HeLa cells, only SETα was recognized by anti-me3-SPK (Fig. 3c). In addition, SETα-FLAG was expressed in Hela cells, immunoprecipitated, and analyzed by MS. The protein was 96.5% trimethylated on its N-terminal Ser (Fig. 3d and Supplementary Fig. S2b). Similar to RCC13, SETα-FLAG with a Gln4 mutation was mostly unmodified, with only 4% mono-methylation, further confirming the importance of Lys4 (Fig. 3d). Finally, a band corresponding to methylated SET was reduced in cells expressing shRNA or siRNA against NRMT (Fig. 1d and Supplementary Fig. S3b).
We next used the anti-me3-SPK antibody, cross-linked to Protein A/G agarose, to immunoprecipitate proteins from mouse spleen and cardiac lysates. Multiple methylated bands were detected (Fig. 3e), and six N-terminally methylated proteins were identified by MS (Fig. 3f). Two, RCC1 and SET, confirm the validity of the approach (Supplementary Fig. S6). The other four - kelch-like protein 3118; (Supplementary Fig. S4c), ribosomal protein L23a, myosin light chain 2, and myosin light chain 3 (Supplementary Figs. S7 and S8) – confirm the predictive power of the identified motif, and expand the verified NRMT substrates.
Among the predicted substrates was the tumor suppressor protein RB. Given the importance of this protein in cell cycle control, we asked whether it too is a bona fide NRMT substrate. First, recombinant NRMT was able to methylate the N-terminal tails of both SETα and RB in vitro (Fig 3g). Second, endogenous RB immunoprecipitated from the colon cancer line HCT116, which has high levels of functional RB19, was recognized by anti-me2-PPK (Fig. 3h). Third, when NRMT was depleted from HCT116 cells, RB methylation was substantially reduced (Fig. 3h and Supplementary Fig. S3g). Surprisingly, RB from HeLa cells, which is inactivated by the E7 oncoprotein20, 21, was not methylated (Fig. 3h), even though HeLa cells express NRMT and contain methylated RCC1 and SET.
Non-methylatable mutants of RCC1 are defective in chromatin association, and their expression in a wild-type background produces supernumerary spindle poles and mis-segregation of mitotic chromosomes, most likely due to the disruption of the Ran gradient3, 11. To test directly the importance of RCC1 α-N-methylation for its function, we used stably silenced NRMT expression in 293LT cells (Fig. 1d), and examined the distribution of RCC1 during mitosis. RCC1 was significantly more diffuse in mitotic cells lacking NRMT than in control cells (Fig. 4a). The mean chromatin/cytoplasm intensity ratio of RCC1 immunofluorescence was ~2-fold greater in the control cells (Fig. 4a). This redistribution could also be observed in living 293LT cells depleted of NRMT and expressing RCC1–RFP (Fig. 4b).
Strikingly, NRMT-depleted 293LT cells in mitosis exhibited >3x more supernumerary spindles than the control (Fig. 4c). These data, together with our previous studies3, argue that one essential role for methylation RCC1 is to stabilize chromatin association, and that this association is necessary for proper mitotic division. However, neither silencing of NRMT nor mutation of the methylation motif significantly altered RB nuclear localization or intranuclear dynamics in HCT116 cells (Supplementary Fig. S9), indicating that the function of the α-N-methylation is not solely to stabilize chromatin associations, but may have a more general role in the regulation of electrostatic interactions.
This study identifies the long-sought eukaryotic α-N-methyltransferase as a conserved member of a superfamily of non-SET domain enzymes and verifies 6 new protein targets. It is likely that NRMT orthologues throughout the Eukarya possess the same specificity and function, because recent screens have detected N-terminal dimethylation of the Arabidopsis and yeast L12 proteins, which possess the same NRMT consensus motif (PPK) as is found in almost all other eukaryotes5, 22. Further analysis of L12 and other target proteins will likely reveal multiple functions for α-N-methylation.
Substrate proteins were expressed with C-terminal His6 tags, and assayed in 50mM Tris, 50mM K Acetate, pH 8.0, with 100 µM SAM as the methyl donor. All reactions were incubated 1 h at 30°C and analyzed for methylation by immunoblot. For ELISA assays, substrates and enzyme were mixed with 0.55 µCi 3H-SAM. Reactions were incubated as above then transferred to Protein A-coated ELISA plates pretreated with anti-me2-SPK. After 1 h, the reaction was removed and wells were treated with 1% SDS for 30 min. This solution was then quantified for incorporated 3H-methyl by scintillation counting.
Efficient removal of the initiating Met is dependent on the identity of the second residue. To avoid this problem, we created fusions from residues 2–10 of RCC1 with His6-tag plus Factor X cleavage site at the N-terminus, and C-terminal GFP. Cleavage with Factor X exposes the second residue of the RCC1 N-terminus. XPK-EGFP substrate proteins were expressed in E.coli and purified on Ni-NTA beads, then cleaved using Factor X.
On-bead endoproteinase Glu-C and Asp-N digestions were used to identify the immunoprecipitated proteins. The retained N-termini were then eluted from the beads and analyzed for methylation (Supplementary Fig. S5).
This work was supported by research grants from the National Institutes of Health to I.G.M., D.F.M, and W.M. C.S.T. was the recipient of a Post-doctoral fellowship from the National Institutes of Health.
Full Methods and associated references are available in the online version of the paper at www.nature.com/nature.
Author Contributions C.S.T. purified NRMT and performed the experiments characterizing its function and targets and wrote the paper with I.G.M. J.J.P. performed the ITC experiments and the RCC1 mutagenesis, and the docking experiments with the aid of M.S. T.M.S. and J.L.B. performed the mass spectrometry with J.S. D.F.H directed the mass spectrometry. W.M. coordinated the experiments of J.P. I.G.M directed the biochemical and cell biological studies.