Protein arginine methyltransferase 6 (PRMT6) catalyses the asymmetric dimethylation of arginines on numerous substrate proteins within the human cell. In particular, PRMT6 methylates histone H3 arginine 2 (H3R2) which affects both gene repression and activation. However, the substrate specificity of PRMT6 has not been comprehensively analysed. Here, we systematically characterise the substrate recognition motif of PRMT6, finding that it has broad specificity and recognises the RG motif. Working with a H3 tail peptide as a template, on which we made 204 amino acid substitutions, we use targeted mass spectrometry to measure their effect on PRMT6 in vitro activity. We first show that PRMT6 methylates R2 and R8 in the H3 peptide, although H3R8 is methylated with lower efficiency and is not an in vivo PRMT6 substrate. We then quantify the effect of 194 of these amino acid substitutions on methylation at both H3R2 and H3R8. In both cases, we find that PRMT6 tolerates essentially any amino acid substitution in the H3 peptide, but that positively charged and bulky residues are preferred near the target arginine. We show that PRMT6 also has preference for glycine, but only in the position immediately following the target arginine. This indicates that PRMT6 recognises the RG motif rather than the RGG motif. We further confirm this preference for the RG motif on another PRMT6 substrate, histone H4R3. This broad specificity and recognition of RG rather than RGG are distinctive among the PRMT family and has implications for the development of drugs to selectively target PRMT6.
Databases: Panorama Public (https://panoramaweb.org/PRMT6motif.url); ProteomeXchange (PXD016711).
Bibliographical noteFunding Information:
The authors acknowledge A/Prof. Mark Raftery and Dr. Ling Zhong for their maintenance of the Orbitrap mass spectrometers housed at the Bioanalytical Mass Spectrometry Facility within the Mark Wainwright Analytical Centre of the University of New South Wales. This work was funded by Australian Research Council grant DP170100108 to MRW and GHS, by DFG (Deutsche Forschungsgemeinschaft) grants (TRR81 A03, BA 2292/1 and KFO325 BA 2292/5) to UMB and by DFG (Deutsche Forschungsgemeinschaft) grant TRR81 Z01 to PK.
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- histone methylation
- protein arginine methyltransferase
- substrate specificity