Protein arginine methylation is a widespread post-translational modification in mammalian cells with suggested regulatory roles in cellular functions such as transcription, translation, cell signaling, DNA repair and protein stability. Protein arginine methyltransferases (PRMTs) modify peptidyl arginine residues by transferring methyl groups from S-adenosyl-L-methionine onto the ω-nitrogen atoms of the guanidino group of the arginine side chain. To date, nine PRMTs have been identified in mammals, seven of their discoveries associated with our laboratory. The PRMTs are classified as type I (PRMTs 1-4, 6, 8), type II (PRMTs 5 and 9), or type III (PRMT7) based on whether they form asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), or ω-monomethylarginine (MMA), respectively.

We have continued to characterize PRMTs, particularly PRMT5, PRMT7, and PRMT9. These enzymes are important in cell proliferation, and excess activities have been associated with cancer. We have recently collaborated with medicinal chemists to help develop inhibitors of these enzymes that may lead to cancer therapeutics.

Publication: Feoli, A., Iannelli, G., Cipriano, A., Milite, C., Shen, L., Wang, Z., Hadjikryiacou, A., Lowe, T. L., Safaeipour, C., Viviano, M., Sarno, G., Morretta, E., Monti, M., Yang, Y., Clarke, S. G., Cosconati, S., Castellano, S., and Sbardella, G. (2023) “Identification of a Protein Arginine Methyltransferase 7 (PRMT7)/Protein Arginine Methyltransferase 9 (PRMT9) Inhibitor” J. Med. Chem. 66, 13665-13683. Selected for cover illustrative art. (DOI: 10.1021/acs.jmedchem.3c01030).

PRMT7 is unique in the family of nine PRMTs in that it only forms the monomethyl MMA derivative. It has a tight specificity for R-X-R sequences, and it is most active under physiological stress conditions. However, there is still a very large knowledge gap in our understanding the physiological function of PRMT7. A central question to be addressed is whether MMA is simply a residue required for normal protein function or whether it represents a reversible modification that modulates protein function. Recently, there has been much interest in identifying the physiological substrates of PRMT7. Multiple in vitro studies suggest that core histones are good substrates of PRMT7, of which histones H2B and H4 are particularly good methyl acceptors. To better understand the specificity of PRMT7 toward basic RXR-containing regions that could be helpful in identifying additional in vivo substrates we explored how sequence substitutions in the 29-RKRSR-33 target sequence in human H2B affect PRMT7 methylation activity and identified motifs that promote methylation of histone H2B.

Publication: Bondoc, T. J., Lowe, T. L., and Clarke, S. G. (2023) ”The exquisite specificity of human protein arginine methyltransferase 7 (PRMT7) toward Arg-X-Arg sites” PLoS ONE 18(5): e0285812. (https://doi.org/10.1371/journal.pone.0285812). PMCID: PMC10202292.

In October of 2024 I submitted a new research proposal to the National Science Foundation – “PPM – How Is Protein Arginine Methylation Coordinated with Other Modifications to Effect Biological Function”. This proposal was written in response to a “Dear Colleague Letter: Posttranscriptional and Posttranslational Modification (PPM)” from the NSF sent out in May of 2024 (NSF 24-084) to solicit applications in an area that my research has been active in. The NSF letter stated that “While modifications of a few protein classes, such as kinases and histones, have been studied extensively, much less is understood about the PTMs of many other proteins. Moreover, how multiple modifications on individual proteins interact to impact assembly, structure and function of complexes and proteomes is also largely unexplored.” The bottom line of the letter was that “the challenge now is to understand how these modifications interact with one another and create multi-layered, dynamic, regulatory networks within cells, that govern organismal phenotype.”

This challenge was right up our alley. Our laboratory had just concluded a very successful collaboration with Professor Margot Quinlan’s laboratory on the interaction of methylation and phosphorylation reactions in controlling the activity of a formin homology domain protein involved in the assembly of actin filaments in cardiac muscle formation. This collaboration led to the publication in October of 2024 of our paper in the Journal of Biological Chemistry entitled “Methylation and phosphorylation of formin homology domain proteins (Fhod1 and Fhod3) by protein argininemethyltransferase 7 (PRMT7) and Rho kinase (ROCK1)” that positioned us well for the NSF solicitation. The proposal was received by the NSF panel and outside reviewers with some enthusiasm and was funded in February of 2026.

Publication: Lowe, T. L., Valencia, D. A., Velasquez, V. E., Quinlan, M. E., and Clarke, S. G. (2024) “Methylation and phosphorylation of formin homology domain proteins (Fhod1 and Fhod3) by protein arginine methyltransferase 7 (PRMT7) and Rho Kinase (ROCK1)” J. Biol. Chem. 300, article 107857, pp. 1-15. (DOI: 10.1016/j.jbc.2024.107857). PMID: 39368550. PMCID: PMC11584945.

We will now study the PRMT7 modification of histone H2B. Histones are the poster children of crosstalk in protein modification, with demonstrated arginine and lysine methylation, lysine acetylation, lysine ubiquitination, and serine and threonine phosphorylation. These modifications make up the “histone code” that determine which genes are activated and which genes are repressed. We will focus on the relationship between PRMT7-dependent modification of histone H2B and a nearby spontaneous modification of an aspartic acid residue. We will determine the stoichiometry of methylation of histone H2B in intact cells to distinguish between regulatory roles and structural roles in protein function.

We will ask whether the activity of PRMT7 is regulated. While there is clear evidence for regulatory mechanisms of many protein kinases and other types of modification enzymes, there is little known about how the activity of PRMTs may be regulated. We propose experiments to identify in vivo substrates of PRMT7 in mammalian cultured cells and determine their stoichiometry under various stress conditions. We would be particularly interested in identifying small molecule or protein activators of PRMT7. I believe that many researchers in this field may be relying on the wrong paradigm (of regulatory control) to understand protein arginine methylation, and the results of our new studies will hopefully open their eyes to the possibility that methylated arginine residues represent amino acid residues with new properties that give additional interactions to protein species.