Identification of substrates and sequence requirements for CARNMT1-mediated histidine methylation of C3H zinc fingers.

Abstract

It has recently become clear that protein histidine methylation is widespread and functionally important, and human CARNMT1 was recently reported as a novel protein histidine methyltransferase (HMT). We describe our independent uncovering of CARNMT1's protein HMT activity, and a comprehensive assessment of its methylation targets and substrate specificity. Using a combination of in vitro methylation of cellular extracts and protein mass spectrometry, we identified several CARNMT1 targets that were fully methylated in cells, all of which were C3H zinc finger (ZnF) proteins. These included the previously identified U2AF1, ZC3H15 and ZC3H18, but also the unreported RBM22, PPP1R10, PRR3 and RNF113A. Using peptide arrays, we investigated CARNMT1-mediated methylation of 145 candidate sequences, encompassing all C3H ZnFs and selected non-ZnFs. We found that only ∼30 % of the tested sequences were methylated, with C3H ZnFs constituting the vast majority of the strongly methylated ones, most of which are also methylated in cells. This establishes peptide methylation as a good predictor of in vivo methylation. To investigate the specificity of CARNMT1, we systematically substituted His-proximal residues in four different substrate peptides. This generated four rather different activity profiles, which were still quite restrictive for each peptide, indicating that molecular recognition by CARNMT1 is context-dependent, and that sequence-based prediction of additional CARNMT1 substrates may be challenging. We also identified several homologous methylation events in Caenorhabditis elegans and showed that they could be introduced by nematode CARNMT in vitro. Thus, CARNMT1 is an evolutionary conserved protein HMT with a complex mode of substrate recognition.