Systematic investigation of PRMT6 substrate recognition reveals broad specificity with a preference for an RG motif or basic and bulky residues.

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).

Assaying epigenome functions of PRMTs and their substrates.

Among the widespread and increasing number of identified post-translational modifications (PTMs), arginine methylation is catalyzed by the protein arginine methyltransferases (PRMTs) and regulates fundamental processes in cells, such as gene regulation, RNA processing, translation, and signal transduction. As epigenetic regulators, PRMTs play key roles in pluripotency, differentiation, proliferation, survival, and apoptosis, which are essential biological programs leading to development, adult homeostasis but also pathological conditions including cancer. A full understanding of the molecular mechanisms that underlie PRMT-mediated gene regulation requires the genome wide mapping of each player, i.e., PRMTs, their substrates and epigenetic marks, methyl-marks readers as well as interaction partners, in a thorough and unambiguous manner. However, despite the tremendous advances in high throughput sequencing technologies and the numerous efforts from the scientific community, the epigenomic profiling of PRMTs as well as their histone and non-histone substrates still remains a big challenge owing to obvious limitations in tools and methodologies. This review will summarize the present knowledge about the genome wide mapping of PRMTs and their substrates as well as the technical approaches currently in use. The limitations and pitfalls of the technical tools along with conventional approaches will be then discussed in detail. Finally, potential new strategies for chromatin profiling of PRMTs and histone substrates will be proposed and described.

Identification and in silico structural analysis of Gallus gallus protein arginine methyltransferase 4 (PRMT4).

Protein arginine methyltransferase 4 (PRMT4) is an essential epigenetic regulator of fundamental and conserved processes during vertebrate development, such as pluripotency and differentiation. Surprisingly, PRMT4 homologs have been identified in nearly all vertebrate classes except the avian genome. This raises the possibility that in birds PRMT4 functions are taken over by other PRMT family members. Here, we reveal the existence of a bona fide PRMT4 homolog in the chicken, Gallus gallus. Using a biochemical approach, we initially purified a putative chicken PRMT4 protein and thus provided the first evidence for the presence of an endogenous PRMT4-specific enzymatic activity toward histone H3 arginine 17 (H3R17) in avian cells. We then isolated a G. gallus PRMT4 (ggPRMT4) transcript encompassing the complete open reading frame. Recombinant ggPRMT4 possesses intrinsic methyltransferase activity toward H3R17. CRISPR/Cas9-mediated deletion of ggPRMT4 demonstrated that the transcript identified here encodes avian PRMT4. Combining protein-protein docking and homology modeling based on published crystal structures of murine PRMT4, we found a strong structural similarity of the catalytic core domain between chicken and mammalian PRMT4. Strikingly, in silico structural comparison of the N-terminal Pleckstrin homology (PH) domain of avian and murine PRMT4 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 PRMT4.

A colorimetric-based amplification system for proteinases including MMP2 and ADAM8.

We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads. The amount of product is then quantified using a standard HRP color development assay employing 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). This HRP amplification system represents a new approach to proteinase assays and could be applied to other enzymes, such as lipases, esterases, and kinases, as long as the unreacted substrate can be physically separated from the product and catalysis by the enzyme to be quantified is not impaired dramatically by steric hindrance from the HRP entity.