Credit: GETTY

Although the post-translational methylation of α-amino groups (α-N-methylation) was discovered over 30 years ago, little was known about its mechanism and function. Indeed, the only known function of α-N-methylation is its requirement by the Ran guanine nucleotide exchange factor RCC1 for normal cell mitosis. Macara and colleagues now describe the first eukaryotic α-N-methyltransferase, named N-terminal RCC1 methyltransferase (NRMT) for its ability to modify RCC1, and identify several new α-N-methylation targets as a result of this discovery.

“NRMT is an α-N-methyltransferase for RCC1.”

The authors assessed HeLa cell nuclear fractions for protein methylation and analysed the fraction that methylated RCC1 using mass spectrometry. They identified an uncharacterized methyltransferase called METTL11a, a member of the methyltransferase 11 family, which they rename NRMT. Overexpression of NRMT in cells increases RCC1 α-N-methylation on the N-terminal Ser, whereas depleting NRMT decreases this, confirming that NRMT is an α-N-methyltransferase for RCC1. The authors sought to gain insight into the function of this modification by examining the distribution of RCC1 in NRMT-depleted cells. Less RCC1 is associated with chromatin in cells lacking NRMT, and NRMT-depleted cells in mitosis exhibit 3 times more supernumerary spindles than controls. These data suggest that RCC1 α-N-methylation stabilizes its association with chromatin to ensure proper mitotic division.

The structure of NRMT in complex with S-adenosylhomocysteine (SAH; which results from the transfer of the methyl group from S-adenosylmethionine to small molecules) was resolved at 1.75 Å by the Structural Genomics Consortium. This revealed a large cavity opposite the SAH-binding site that can accommodate N-terminal peptides. Using computer software the authors modelled an RCC1 N-terminal peptide (Ser-Pro-Lys-Arg-Ile-Ala) in the NRMT putative active site, and this suggested that only the first three residues of this motif interact with NRMT. Indeed, mutational analyses confirm that the motif X-Pro-Lys is important for substrate recognition by NRMT.

So, can the NRMT recognition sequence be used to identify new targets of α-N-methylation? The authors searched GenBank for NRMT candidate substrates using Met-(Ala/Ser/Pro)-Pro-Lys and also screened mouse tissues for α-N-methylation targets using methylation-specific antibodies. Notable predicted substrates from these analyses include the nuclear oncogene SET (also known as TAFI and PHAPII) and the tumour suppressor retinoblastoma (RB). Recombinant NRMT can methylate the N-terminal tail of both SET and RB in vitro. Furthermore, when NRMT is depleted from cells, SET and RB methylation is substantially reduced. Thus, SET and RB are novel α-N-methylated proteins and NRMT is the enzyme that catalyzes this modification.

In short, this study identifies NRMT as the eukaryotic α-N-methyltransferase and uses this knowledge to provide further insight into the function of RCC1 α-N-methylation and to identify two new α-N-methylation targets, and more than 35 putative targets, for further analysis.