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.

Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that modifies histone tails. To help elucidate the biological function of PRMT6 in vivo, we generated transgenic mice that ubiquitously express PRMT6 fused to the hormone-binding portion of the estrogen receptor (ER*). The ER*-PRMT6 fusion is unstable and cytoplasmic, but upon systemic treatment with tamoxifen, it becomes stabilized and translocates into the nucleus. As a result, a dramatic increase in the H3R2me2a histone mark is observed. We found that one consequence of induced ER*-PRMT6 activation is increased IL-6 levels. IL-6 expression is regulated by the nuclear factor-kappa B (NF-κB) transcription factor, and PRMT6 functions as a coactivator of this pathway. We show that PRMT6 directly interacts with RelA, and that its overexpression enhances the transcriptional activity of an ectopic NF-κB reporter and endogenously regulates NF-κB target genes. PRMT6 is recruited, by RelA, to selective NF-κB target promoters upon TNF-α stimulation. Moreover, ER*-PRMT6 activation causes RelA accumulation in the nucleus. In summary, we observe that PRMT6 is recruited to chromatin at selective NF-κB target promoters, where it likely impacts the histone code and/or methylates other chromatin-associated proteins to facilitate transcription.

Previous studies suggested that mdivi-1 (mitochondrial division inhibitor), a putative inhibitor of dynamin-related protein (DRP1), decreased cancer cell proliferation through inducing mitochondrial fusion and altering oxygen consumption. However, the metabolic reprogramming underlying the DRP1 inhibition is still unclear in cancer cells.

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.

The Tudor domain-containing proteins are characterized by their specific interactions with methylated protein motifs, including methyl-arginines and methyl-lysines. The Tudor domain-containing protein 3 (TDRD3) is one of the major methyl-arginine effector molecules that recognizes methylated arginine residues on histones and the C-terminal domain of RNA polymerase II, and activates transcription. However, majority of the cellular TDRD3 localizes to the cytoplasm and its functions there are still elusive. Here, we have identified ubiquitin-specific protease 9 X-linked (USP9X) as a TDRD3-interacting protein by GST (glutathione S-transferase) pull-down and co-immunoprecipitation. Detailed characterization suggests that the interaction between TDRD3 and USP9X is mediated through the Tudor domain of TDRD3 and the arginine methylation of USP9X. This interaction plays a critical role in TDRD3 protein stability, as knockdown of USP9X expression leads to increased TDRD3 ubiquitination. We also found that USP9X co-localizes with TDRD3 in cytoplasmic stress granules and this localization is diminished in Tdrd3-null mouse embryonic fibroblast cells, suggesting that TDRD3 is essential for USP9X stress granule localization. Furthermore, we found that one of the USP9X de-ubiquitination targets, myeloid cell leukemia protein 1, is regulated by TDRD3, indicating that TDRD3 potentially regulates USP9X de-ubiquitinase activity. Finally, we show that knockdown of TDRD3 expression sensitizes breast cancer cells to chemotherapeutic drug-induced apoptosis, likely due to its regulation of USP9X. This study provides a novel candidate strategy for targeting apoptosis pathways in cancer therapy.

R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic 'reader' Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.

The coactivator-associated arginine methyltransferase (CARM1) functions as a regulator of transcription by methylating a diverse array of substrates. To broaden our understanding of CARM1's mechanistic actions, we sought to identify additional substrates for this enzyme. To do this, we generated CARM1 substrate motif antibodies, and used immunoprecipitation coupled with mass spectrometry to identify cellular targets of CARM1, including mediator complex subunit 12 (MED12) and the lysine methyltransferase KMT2D. Both of these proteins are implicated in enhancer function. We identified the major CARM1-mediated MED12 methylation site as arginine 1899 (R1899), which interacts with the Tudor domain-containing effector molecule, TDRD3. Chromatin immunoprecipitation-seq studies revealed that CARM1 and the methyl mark it deposits are tightly associated with ERα-specific enhancers and positively modulate transcription of estrogen-regulated genes. In addition, we showed that the methylation of MED12, at the R1899 site, and the recruitment of TDRD3 by this methylated motif are critical for the ability of MED12 to interact with activating noncoding RNAs.

The arginine methylation status of histones dynamically changes during many cellular processes, including hematopoietic stem/progenitor cell (HSPC) development. The arginine methyltransferases and the readers that transduce the histone codes have been defined. However, whether arginine demethylation actively occurs in cells and what enzyme demethylates the methylarginine residues during various cellular processes are unknown. We report that JMJD1B, previously identified as a lysine demethylase for H3K9me2, mediates arginine demethylation of H4R3me2s and its intermediate, H4R3me1. We show that demethylation of H4R3me2s and H3K9me2s in promoter regions is correlated with active gene expression. Furthermore, knockout of JMJD1B blocks demethylation of H4R3me2s and/or H3K9me2 at distinct clusters of genes and impairs the activation of genes important for HSPC differentiation and development. Consequently, JMJD1B-/- mice show defects in hematopoiesis. Altogether, our study demonstrates that demethylase-mediated active arginine demethylation process exists in eukaryotes and that JMJD1B demethylates both H4R3me2s and H3K9me2 for epigenetic programming during hematopoiesis.

DNA topoisomerase 3B (TOP3B) is unique among all mammalian topoisomerases for its dual activities that resolve both DNA and RNA topological entanglements to facilitate transcription and translation. However, the mechanism by which TOP3B activity is regulated is still elusive. Here, we have identified arginine methylation as an important post-translational modification (PTM) for TOP3B activity. Protein arginine methyltransferase (PRMT) 1, PRMT3 and PRMT6 all methylate TOP3B in vitro at its C-terminal arginine (R) and glycine (G)-rich motif. Site-directed mutagenesis analysis identified R833 and R835 as the major methylation sites. Using a methylation-specific antibody, we confirmed that TOP3B is methylated in cells and that mutation of R833 and R835 to lysine (K) significantly reduces TOP3B methylation. The methylation-deficient TOP3B (R833/835K) is less active in resolving negatively supercoiled DNA, which consequently lead to accumulation of co-transcriptionally formed R-loops in vitro and in cells. Additionally, the methylation-deficient TOP3B (R833/835K) shows reduced stress granule localization, indicating that methylation is critical for TOP3B function in translation regulation. Mechanistically, we found that R833/835 methylation is partially involved in the interaction of TOP3B with its auxiliary factor, the Tudor domain-containing protein 3 (TDRD3). Together, our findings provide the first evidence for the regulation of TOP3B activity by PTM.