Figure 1. NRMT1 methylates CENP-A in vitro and in vivo.

(a) The initiating Methionine of CENP-A is removed by methionine aminopeptidase. Posttranslational modifications of the amino terminus are indicated: Me, α-amino-terminal methylation; P, phosphorylation. (b) Schematic of the first 10 amino acids of the CENP-A tail fused with GFP on the C-terminus and 6XHis on the N-terminus. Factor X cleavage exposes the N-terminal glycine for modification by NRMT. (c) Anti-me3-CENP-A antibody specifically recognizes CENP-A methylated in vitro by NRMT (d) Western blot of extracts from HeLa cells stably expressing CENP-A-eGFP in which NRMT was suppressed by shRNA shows a loss of CENP-A α-amino trimethylation. (e) Immunofluorescence analysis of the HeLa cell treated with NRMT1 shRNA using CENP-A me3 antibody shows loss of CENP-A methylation at centromeres. (f) Immunofluorescence using CENP-A me3 antibody of HeLa cell stably expressing CENP-A-LAP and treated with NRMT1 shRNA. Scale bar, 10 μm. Error bars indicate s.d. Experiment done in duplicates. (g) Amino acid sequence of the CENP-A mutants used in this study. (h) In vitro NRMT1 methylation assay using factor X cleaved CENP-A tail as a substrate in the presence of ³H-S-adenosyl-methionine. A filter-binding assay was used to determine the incorporation of radioactive methyl groups into CENP-A amino termini. The experiment was done in triplicate, n=3. Error bars indicate s.d. (i,j) Immunoblot using the methyl specific CENP-A antibody of extracts from cells expressing CENP-A S16 and S18 phosphorylation mutants show that phosphorylation of S16 and S18 are not required for methylation of CENP-A. Likewise, mutations that eliminate CENP-A methylation do not affect the phosphorylation of CENP-A as shown by immunoblot with a phosphospecific CENP-A antibody.