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

Abstract

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.

Protein arginine methyltransferases: insights into the enzyme structure and mechanism at the atomic level

“…Over 130 PRMT4 protein substrates have been identified and most of them contain proline-rich motifs [78]. Deletion of the PH domain of PRMT4 results in a global decrease in both the interaction of the PRMT4 with its substrates and methylation activities [78,79]. Interestingly, structural studies of PRMT4 revealed striking differences in the orientation of the N terminus upon AdoHcy binding (Fig.…”

PRMT1 promotes the tumor suppressor function of p14 ARF and is indicative for pancreatic cancer prognosis

“…In vitro methylation assays were performed in the presence of radiolabeled SAM ( 14 C‐labeld methyl‐group) using recombinant GST‐tagged substrates and PRMT enzymes purified from bacteria or eukaryotic cells as described in (Hyllus et al, 2007; Berberich et al, 2017). Eluted GST‐tagged or Flag‐tagged PRMT enzymes or immunoprecipitated HA‐tagged PRMT5/Myc‐tagged MEP50 were incubated with substrates (either bead‐bound GST‐tagged proteins or histone H3, H4, or bulk histones from calf thymus; Sigma‐Aldrich) in the presence of [ 14 C‐methyl]‐S‐adenosyl‐methionine ( 14 C‐methyl‐SAM 20 µCi/ml; Perkin Elmer) for 1–2 h at 37°C.…”

“…Cell lines and reagents U2OS, HeLa, HEK293T, PaTu8988t, MiaPaCa-2, S2-007, and Panc1 cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS, Gibco/BRL), 1% penicillin/streptomycin at 37°C and 5% CO 2 . Sf9 cells were cultured as described in (Berberich et al, 2017). Detailed information on cell culture reagents, manipulations, plasmids, and antibodies (including the generation of a-me-p14 ARF ) is supplied in the Appendix Supplementary Methods.…”

CRISPR-Cas9-mediated chicken prmt5 gene knockout and its critical role in interferon regulation

“…In chicken, PRMT1, PRMT4, PRMT5, and PRMT8 have recently been identified ( Berberich et al, 2017 ; Wang et al, 2017 ; Hu et al, 2023a , b ); however, PRMT2 and PRMT6 seem absent in chickens. As known, PRMT5 regulates the production of type I interferons ( IFNs ), impairing the host defenses against RNA/DNA virus infection in human ( Cui et al, 2020 ), but the role of chicken PRMT5 in the innate immune response remains unclear.…”