Caenorhabditis elegans PRMT-7 and PRMT-9 Are Evolutionarily Conserved Protein Arginine Methyltransferases with Distinct Substrate Specificities.

Caenorhabditis elegans protein arginine methyltransferases PRMT-7 and PRMT-9 are two evolutionarily conserved enzymes, with distinct orthologs in plants, invertebrates, and vertebrates. Biochemical characterization of these two enzymes reveals that they share much in common with their mammalian orthologs. C. elegans PRMT-7 produces only monomethylarginine (MMA) and preferentially methylates R-X-R motifs in a broad collection of substrates, including human histone peptides and RG-rich peptides. In addition, the activity of the PRMT-7 enzyme is dependent on temperature, the presence of metal ions, and the reducing agent dithiothreitol. C. elegans PRMT-7 has a substrate specificity and a substrate preference different from those of mammalian PRMT7, and the available X-ray crystal structures of the PRMT7 orthologs show differences in active site architecture. C. elegans PRMT-9, on the other hand, produces symmetric dimethylarginine and MMA on SFTB-2, the conserved C. elegans ortholog of human RNA splicing factor SF3B2, indicating a possible role in the regulation of nematode splicing. In contrast to PRMT-7, C. elegans PRMT-9 appears to be biochemically indistinguishable from its human ortholog.

Protein Arginine Methyltransferase Product Specificity Is Mediated by Distinct Active-site Architectures.

In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors.

Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2.

Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.

Substrate specificity of human protein arginine methyltransferase 7 (PRMT7): the importance of acidic residues in the double E loop.

Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion protein with a broad range of peptide and protein substrates. After confirming its type III activity generating only ω-N(G)-monomethylarginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease.

Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and arginine-rich regions.

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.

Enhancing the Small-Scale Screenable Biological Space beyond Known Chemogenomics Libraries with Gray Chemical Matter─Compounds with Novel Mechanisms from High-Throughput Screening Profiles.

Phenotypic assays have become an established approach to drug discovery. Greater disease relevance is often achieved through cellular models with increased complexity and more detailed readouts, such as gene expression or advanced imaging. However, the intricate nature and cost of these assays impose limitations on their screening capacity, often restricting screens to well-characterized small compound sets such as chemogenomics libraries. Here, we outline a cheminformatics approach to identify a small set of compounds with likely novel mechanisms of action (MoAs), expanding the MoA search space for throughput limited phenotypic assays. Our approach is based on mining existing large-scale, phenotypic high-throughput screening (HTS) data. It enables the identification of chemotypes that exhibit selectivity across multiple cell-based assays, which are characterized by persistent and broad structure activity relationships (SAR). We validate the effectiveness of our approach in broad cellular profiling assays (Cell Painting, DRUG-seq, and Promotor Signature Profiling) and chemical proteomics experiments. These experiments revealed that the compounds behave similarly to known chemogenetic libraries, but with a notable bias toward novel protein targets. To foster collaboration and advance research in this area, we have curated a public set of such compounds based on the PubChem BioAssay dataset and made it available for use by the scientific community.

Identification of a Protein Arginine Methyltransferase 7 (PRMT7)/Protein Arginine Methyltransferase 9 (PRMT9) Inhibitor.

Less studied than the other protein arginine methyltransferase isoforms, PRMT7 and PRMT9 have recently been identified as important therapeutic targets. Yet, most of their biological roles and functions are still to be defined, as well as the structural requirements that could drive the identification of selective modulators of their activity. We recently described the structural requirements that led to the identification of potent and selective PRMT4 inhibitors spanning both the substrate and the cosubstrate pockets. The reanalysis of the data suggested a PRMT7 preferential binding for shorter derivatives and prompted us to extend these structural studies to PRMT9. Here, we report the identification of the first potent PRMT7/9 inhibitor and its binding mode to the two PRMT enzymes. Label-free quantification mass spectrometry confirmed significant inhibition of PRMT activity in cells. We also report the setup of an effective AlphaLISA assay to screen small molecule inhibitors of PRMT9.

PRMT9 is a type II methyltransferase that methylates the splicing factor SAP145.

The human genome encodes a family of nine protein arginine methyltransferases (PRMT1-9), whose members can catalyse three distinct types of methylation on arginine residues. Here we identify two spliceosome-associated proteins-SAP145 and SAP49-as PRMT9-binding partners, linking PRMT9 to U2 snRNP maturation. We show that SAP145 is methylated by PRMT9 at arginine 508, which takes the form of monomethylated arginine (MMA) and symmetrically dimethylated arginine (SDMA). PRMT9 thus joins PRMT5 as the only mammalian enzymes capable of depositing the SDMA mark. Methylation of SAP145 on Arg 508 generates a binding site for the Tudor domain of the Survival of Motor Neuron (SMN) protein, and RNA-seq analysis reveals gross splicing changes when PRMT9 levels are attenuated. These results identify PRMT9 as a nonhistone methyltransferase that primes the U2 snRNP for interaction with SMN.