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Results: 4

1.
Figure 4

Figure 4. Silencing NRMT reduces RCC1 association with chromatin, and increases frequency of mitotic defects. From: NRMT is an ?-N-methyltransferase that methylates RCC1 and Retinoblastoma Protein.

a, Lentiviral silencing of NRMT decreases RCC1 association with chromatin in mitotic (arrowheads) 293LT cells. Chromatin to cytoplasmic ratio of endogenous RCC1 is 2x higher in control, compared to NRMT-depleted mitotic cells. Data analyzed by two-tailed independent t tests. n=50 mitotic cells for each condition. Error bars: +/− 1 s.e.m. b, Lentiviral silencing of NRMT decreases the association of RCC1-RFP with chromatin in live 293LT cells. Data were analyzed by two-tailed independent t tests. n=50 mitotic cells for each condition. Error bars: +/− 1 s.e.m. c, Lentiviral silencing of NRMT increases the frequency of supernumerary spindles to 3x higher than that of control cells. Data were analyzed by a χ2 test. n=109 mitotic cells for each condition. Error bars: +/− 1 s.d.

Christine E. Schaner Tooley, et al. Nature. ;466(7310):1125-1128.
2.
Figure 2

Figure 2. Substrate recognition motif of NRMT. From: NRMT is an ?-N-methyltransferase that methylates RCC1 and Retinoblastoma Protein.

a, NRMT structure with distribution of electrostatic potential. Electrostatic potential was calculated using APBS Tools Plug in23 with default settings, visualized with PyMol program (DeLano Scientific LLC) and colored from red (−1 kT) to blue (+1 kT). SPK-RCC1 hexapeptide (SPKRIA) is modeled into the active site. b, Schematic view of the interactions between the RCC1 substrate peptide (light blue) and the conserved residues of NRMT. Interactions between Lys4 of the RCC1 peptide and Asp178, Asp181, and Ser183 of NRMT are shown. All distances are in Å. c, Modeling of RCC1 mutant hexapeptide (yellow), in which Lys4 was replaced by Gln, into NRMT active site. d, Mutational analysis of NRMT residues predicted to be involved in SPK-RCC1 recognition. e, The affinity of the enzyme was measured using isothermal titration calorimetry. Wild-type 12-residue RCC1 N-terminal peptide showed exothermic binding to NRMT (ΔH= −9.8kcal/mol) with a Kd 70µM (left panel). A mutant peptide in which Gln replaced Lys4 showed no detectable binding (right panel).

Christine E. Schaner Tooley, et al. Nature. ;466(7310):1125-1128.
3.
Figure 1

Figure 1. Mettl11a is the α-N-RCC1 methyltransferase (NRMT). From: NRMT is an ?-N-methyltransferase that methylates RCC1 and Retinoblastoma Protein.

a, Purification scheme for RCC1 N-terminal methyltransferase. b, In vitro methylation assay (NE–nuclear extract, D–dialyzed nuclear extract, FT–flow thru, W-wash) showing activity elutes in the 40 and 60 mM NaP fractions. c, ELISA assay showing NRMT over-expression increases RCC1 α-N methylation. Data were analyzed by two-tailed independent t tests. n=3–5 independent reactions per condition. Error bars represent +/− 1 s.d. d, Lentiviral knockdown of NRMT in 293LT cells significantly decreases NRMT (arrowhead), di- and trimethylated RCC1, and levels of another methylated protein, later shown to be SET(*), as compared to control cells. β-catenin was the loading control. e, Expression of murine NRMT-FLAG rescues RCC1 methylation levels in NRMT knockdown cells. f, In vitro methylation assays showing immunoprecipitated FLAG-NRMT methylates RCC1-His6. (1-IP input, 2-Substrate only, 3-FLAG-NRMT Elution, 4-FLAG-NRMT elution without substrate, 5-Control IP elution). g, His6-NRMT di-and trimethylates SPK-RCC1-His6 and dimethylates PPK-RCC1-His6. h, Immunofluorescence of endogenous NRMT in HeLa cells +/− NRMT siRNA.

Christine E. Schaner Tooley, et al. Nature. ;466(7310):1125-1128.
4.
Figure 3

Figure 3. NRMT methylates many targets, including SETα and RB. From: NRMT is an ?-N-methyltransferase that methylates RCC1 and Retinoblastoma Protein.

a, His6-NRMT can methylate RCC1-His6 with any amino acid in the second position except Leu, Ile, Trp, Asp, and Glu. Data were compared to the unmethylatable SPQ mutant by two-tailed independent t tests. * indicates P<0.01. n=3 independent reactions per mutant. Error bars: +/− 1 s.d. b, Coomassie stain of ~40 kD protein immunoprecipitated by anti-me3-SPK (*). c, SETα-GFP is recognized by the anti-me3-SPK antibody. d, MS analysis showing SETα-FLAG is stoichiometrically N-terminally trimethylated in HeLa cells. Mutation of SETα Lys4 to Gln abolishes methylation. e, Immunoprecipitations with anti-me3-SPK from mouse spleen (shown) and heart lysate produce at least 3 specific bands visible by Coomassie staining (*). These bands (*), and one additional (+), are recognized by immunoblotting with anti-me3-SPK and correspond to the sizes of the identified mouse proteins (kelch-like 31=70kD, RCC1=45 kD, SET=33kD, and myosin light chain or ribosomal protein L23a ~20 kD). f, Table of N-terminal sequence and methylation status of identified targets. g, NRMT-His6 can methylate in vitro the N-termini of SET and RB fused to GFP. h, Endogenous RB from HCT116 cells, but not HeLa cells, is recognized by the anti-me2-PPK antibody (left panel) and depletion of NRMT in HCT116 cells substantially reduces RB α-N methylation (right panel).

Christine E. Schaner Tooley, et al. Nature. ;466(7310):1125-1128.

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