There are nine protein arginine methyltransferases (PRMTs) encoded in mammalian genomes, the protein products of which catalyse three types of arginine methylation--monomethylation and two types of dimethylation. Protein arginine methylation is an abundant modification that has been implicated in signal transduction, gene transcription, DNA repair and mRNA splicing, among others. Studies have only recently linked this modification to carcinogenesis and metastasis. Sequencing studies have not generally found alterations to the PRMTs; however, overexpression of these enzymes is often associated with various cancers, which might make some of them viable targets for therapeutic strategies.

SUMMARY Tudor domain containing protein 3 (TDRD3) is a major methyl-arginine effector molecule that “reads” methyl-histone marks and facilitates gene transcription. However, the underlying mechanism by which TDRD3 functions as a transcriptional coactivator is unknown. We identified topoisomerase IIIB (TOP3B) as a component of the TDRD3 complex. TDRD3 serves as a molecular bridge between TOP3B and arginine-methylated histones. The TDRD3-TOP3B complex is recruited to the c-MYC gene promoter primarily by the H4R3me2a mark, and the complex promotes c-MYC gene expression. TOP3B relaxes negative supercoiled DNA and reduces transcription-generated R-loops in vitro. TDRD3 knockdown in cells increases R-loop formation at the c-MYC locus, and Tdrd3-null mice exhibit elevated R-loop formation at this locus in B cells. Tdrd3-null mice show significantly increased c-Myc/Igh translocation, a process driven by R-loop structures. By reducing negative supercoiling and resolving R-loop, TOP3B promotes transcription, protects against DNA damage and reduces the frequency of chromosomal translocations.

The human genome encodes a family of nine protein arginine methyltransferases (PRMT1-9), which members can catalyze 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 U2snRNP 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 Arg508 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 non-histone methyltransferase that primes the U2snRNP for interaction with SMN.

Summary Specific sites of histone tail methylation are associated with transcriptional activity at gene loci. These methyl-marks are interpreted by effector molecules, which harbor protein domains that bind the methylated motifs and facilitate either active or inactive states of transcription. CARM1 and PRMT1 are transcriptional coactivators that deposit H3R17me2a and H4R3me2a marks, respectively. We used a protein domain microarray approach to identify the tudor domain-containing protein TDRD3 as a “reader” of these marks. Importantly, TDRD3 itself is a transcriptional coactivator. This coactivator activity requires an intact tudor domain. TDRD3 is recruited to an estrogen responsive element in a CARM1-dependent manner. Furthermore, ChIP-seq analysis of TDRD3 reveals that it is predominantly localized to transcriptional start sites. Thus, TDRD3 is an effector molecule that promotes transcription by binding methylarginine marks on histone tails.

Background: Newly discovered protein arginine methyltransferase 9 (PRMT9) modulates alternative splicing by methylation of SF3B2. Results: Biochemical probes of PRMT9 and its substrate protein revealed domains and residues required for methylation. Conclusion: PRMT9 is unique among PRMTs in its narrow range of methyl-accepting substrates. Significance: Understanding PRMT9 catalysis will help elucidate how it may control the activity of SF3B2 and other potential endogenous substrates.

Androgen receptor (AR) is essential for the maintenance of the male reproductive systems and is critical for the carcinogenesis of human prostate cancers (PCas). D-type cyclins are closely related to the repression of AR function. It has been well documented that cyclin D1 inhibits AR function through multiple mechanisms, but the mechanism of how cyclin D3 exerts its repressive role in the AR signaling pathway remains to be identified. In the present investigation, we demonstrate that cyclin D3 and the 58-kDa isoform of cyclin-dependent kinase 11 (CDK11 p58 ) repressed AR transcriptional activity as measured by reporter assays of transformed cells and prostate-specific antigen expression in PCa cells. AR, cyclin D3, and CDK11 p58 formed a ternary complex in cells and were colocalized in the luminal epithelial layer of the prostate. AR activity is controlled by phosphorylation at specific sites. We found that AR was phosphorylated at Ser-308 by cyclin D3/CDK11 p58 in vitro and in vivo, leading to the repressed activity of AR transcriptional activation unit 1 (TAU1). Furthermore, androgen-dependent proliferation of PCa cells was inhibited by cyclin D3/CDK11 p58 through AR repression. These data suggest that cyclin D3/CDK11 p58 signaling is involved in the negative regulation of AR function.Androgen receptor (AR), a member of the nuclear receptor family, directly regulates patterns of gene expression in response to the steroids testosterone and dihydrotestosterone (DHT) and is subsequently involved in the regulation of the development and differentiation of the male reproductive system (10). Similar to other steroid receptors, AR contains a transactivation domain (TAD), also named AF-1, in its N terminus, a ligand binding domain (LBD) in its C terminus, a DNA-binding domain (DBD), and a hinge region between the TAD and LBD. The transcriptional activation unit 1 (TAU1) and TAU5 motifs in the AR N-terminal domain (NTD) (residues 101 to 370 and 360 to 485, respectively) as well as the AF-2 motif in the AR LBD have been implicated in directly contacting p160 proteins and mediating transcription (1,23,31,48).AR is a phosphoprotein whose function is regulated by the modulation of its phosphorylation status at different sites (4). The consensus phosphorylation sites found in AR indicate that AR could be a substrate for DNA-dependent kinase, protein kinase A, protein kinase C, mitogen-activated protein kinase, and casein kinase 2 (4). Ser-16, Ser-81, Ser-94, Ser-256, Ser-308, Ser-424, and Ser-650 have been identified as being phosphorylation sites of AR by mutagenesis, peptide mapping, and mass spectrometry (6, 60). Recently, several Ser/Thr protein kinases have been found to phosphorylate AR at the abovementioned sites in vitro and in vivo. For example, AR Ser-515 is phosphorylated by mitogen-activated protein kinase, Ser-213 and Ser-791 are phosphorylated by Akt, and Ser-650 is phosphorylated by p38␣ and JNK1 (17,30,57).More and more studies suggest that cyclins and cyclin-dependent kinases (CDKs) are also involved in th...

The elevated levels of ␤1,4-galactosyltransferase I (GalT I; EC 2.4.1.38) are detected in highly metastatic lung cancer PGBE1 cells compared with its less metastatic partner PGLH7 cells. Decreasing the GalT I surface expression by small interfering RNA or interfering with the surface of GalT I function by mutation inhibited cell adhesion on laminin, the invasive potential in vitro, and tyrosine phosphorylation of focal adhesion kinase. The mechanism by which GalT I activity is up-regulated in highly metastatic cells remains unclear. To investigate the regulation of GalT I expression, we cloned the 5-region flanking the transcription start point of the GalT I gene (؊1653 to ؉52). Cotransfection of the GalT I promoter/luciferase reporter and the Ets family protein E1AF expression plasmid increased the luciferase reporter activity in a dose-dependent manner. By deletion and mutation analyses, we identified an Ets-binding site between nucleotides ؊205 and ؊200 in the GalT I promoter that was critical for responsiveness to E1AF. It was identified that E1AF could bind to and activate the GalT I promoter by electrophoretic mobility shift assay in PGLH7 cells and COS1 cells. A stronger affinity of E1AF for DNA has contributed to the elevated expression of GalT I in PGBE1 cells. Stable transfection of the E1AF expression plasmid resulted in increased GalT I expression in PGLH7 cells, and stable transfectants migrated faster than control cells. Meanwhile, the content of the ␤1,4-Gal branch on the cell surface was increased in stably transfected PGLH7 cells. GalT I expression can also be induced by epidermal growth factor and dominant active Ras, JNK1, and ERK1. These data suggest an essential role for E1AF in the activation of the human GalT I gene in highly metastatic lung cancer cells.The enzyme ␤1,4-galactosyltransferase I (GalT I 1 ; EC 2.4.1.38) is a constitutively expressed type II membrane-bound glycoprotein in vertebrates (1). It is unusual that it resides in two distinct subcellular compartments, the trans-Golgi network and the cell surface (2, 3). In the trans-Golgi complex, GalT I is one of the key enzymes involved in the sugar chain synthesis that catalyzes the transfer of galactose from UDP-Gal to terminal N-acetylglucosamine, forming the Gal␤134GlcNAc structure (4). Cell surface GalT I acts as a recognition molecule and participates in a number of cellular interactions, including neurite extension, cell growth, spermegg interaction, cell spreading, and migration (5-9).Neoplasms undergo various changes in the carbohydrate moieties of their glycoconjugates, which indicate that the glycosyltransferases themselves may change in malignancies. Consistent with this hypothesis, the importance of specific sialyltransferases, fucosyltransferases, N-acetylglucosaminyltransferase in tumorigenesis, and metastasis has been demonstrated (10 -12).Although the precise role of oligosaccharides in metastasis is presently unknown, accumulated evidence has shown that a number of highly metastatic murine and human cell lines are ...

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.