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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.

Protein arginine methyltransferase 4 ( PRMT 4) is an essential epigenetic regulator of fundamental and conserved processes during vertebrate development, such as pluripotency and differentiation. Surprisingly, PRMT 4 homologs have been identified in nearly all vertebrate classes except the avian genome. This raises the possibility that in birds PRMT 4 functions are taken over by other PRMT family members. Here, we reveal the existence of a bona fide PRMT 4 homolog in the chicken, Gallus gallus . Using a biochemical approach, we initially purified a putative chicken PRMT 4 protein and thus provided the first evidence for the presence of an endogenous PRMT 4‐specific enzymatic activity toward histone H3 arginine 17 (H3R17) in avian cells. We then isolated a G. gallus PRMT 4 (gg PRMT 4 ) transcript encompassing the complete open reading frame. Recombinant gg PRMT 4 possesses intrinsic methyltransferase activity toward H3R17. CRISPR /Cas9‐mediated deletion of gg PRMT 4 demonstrated that the transcript identified here encodes avian PRMT 4. Combining protein–protein docking and homology modeling based on published crystal structures of murine PRMT 4, we found a strong structural similarity of the catalytic core domain between chicken and mammalian PRMT 4. Strikingly, in silico structural comparison of the N‐terminal Pleckstrin homology ( PH ) domain of avian and murine PRMT 4 identified strictly conserved amino acids that are involved in an interaction interface toward the catalytic core domain, facilitating for the first time a prediction of the relative spatial arrangement of these two domains. Our novel findings are particularly exciting in light of the essential function of the PH domain in substrate recognition and methylation by PRMT 4.