Alternatively spliced protein arginine methyltransferase 1 isoform PRMT1v2 promotes the survival and invasiveness of breast cancer cells

Protein arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and plays an important role in many cellular processes. Aberrant PRMT expression has been observed in several common cancer types; however, their precise contribution to the cell transformation process is not well understood. We previously reported that the PRMT1 gene generates several alternatively spliced isoforms, and our initial biochemical characterization of these isoforms revealed that they exhibit distinct substrate specificity and subcellular localization. We focus here on the PRMT1v2 isoform, which is the only predominantly cytoplasmic isoform, and we have found that its relative expression is increased in breast cancer cell lines and tumors. Specific depletion of PRMT1v2 using RNA interference caused a significant decrease in cancer cell survival due to an induction of apoptosis. Furthermore, depletion of PRMT1v2 in an aggressive cancer cell line significantly decreased cell invasion. We also demonstrate that PRMT1v2 overexpression in a non-aggressive cancer cell line was sufficient to render them more invasive. Importantly, this novel activity is specific to PRMT1v2, as overexpression of other isoforms did not enhance invasion. Moreover, this activity requires both proper subcellular localization and methylase activity. Lastly, PRMT1v2 overexpression altered cell morphology and reduced cell-cell adhesion, a phenomenon that we convincingly linked with reduced β-catenin protein expression. Overall, we demonstrate a specific role for PRMT1v2 in breast cancer cell survival and invasion, underscoring the importance of identifying and characterizing the distinct functional differences between PRMT1 isoforms.     

Dynamics of human protein arginine methyltransferase 1(PRMT1) in vivo

Arginine methylation is a posttranslational protein modification catalyzed by a family of protein arginine methyltransferases (PRMT), the predominant member of which is PRMT1. Despite its major role in arginine methylation of nuclear proteins, surprisingly little is known about the subcellular localization and dynamics of PRMT1. We show here that only a fraction of PRMT1 is located in the nucleus, but the protein is predominantly cytoplasmic. Fluorescence recovery after photobleaching experiments reveal that PRMT1 is highly mobile both in the cytoplasm and the nucleus. However, inhibition of methylation leads to a significant nuclear accumulation of PRMT1, concomitant with the appearance of an immobile fraction of the protein in the nucleus, but not the cytoplasm. Both the accumulation and immobility of PRMT1 is reversed when re-methylation is allowed, suggesting a mechanism where PRMT1 is trapped by unmethylated substrates such as core histones and heterogeneous nuclear ribonucleoprotein proteins until it has executed the methylation reaction.     

RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex composition and substrate specificity

Protein arginine methylation plays a critical role in differential gene expression through modulating protein-protein and protein-DNA/RNA interactions. Although numerous proteins undergo arginine methylation, only limited information is available on how protein arginine methyltransferases (PRMTs) identify their substrates. The human PRMT5 complex consists of PRMT5, WD45/MEP50 (WD repeat domain 45/methylosome protein 50), and pICln and catalyzes the symmetrical arginine dimethylation of its substrate proteins. pICln recruits the spliceosomal Sm proteins to the PRMT5 complex for methylation, which allows their subsequent loading onto snRNA to form small nuclear ribonucleoproteins. To understand how the PRMT5 complex is regulated, we investigated its biochemical composition and identified RioK1 as a novel, stoichiometric component of the PRMT5 complex. We show that RioK1 and pICln bind to PRMT5 in a mutually exclusive fashion. This results in a PRMT5-WD45/MEP50 core structure that either associates with pICln or RioK1 in distinct complexes. Furthermore, we show that RioK1 functions in analogy to pICln as an adapter protein by recruiting the RNA-binding protein nucleolin to the PRMT5 complex for its symmetrical methylation. The exclusive interaction of PRMT5 with either pICln or RioK1 thus provides the first mechanistic insight into how a methyltransferase can distinguish between its substrate proteins.     

RNA binding protein RALY promotes Protein Arginine Methyltransferase 1 alternatively spliced isoform v2 relative expression and metastatic potential in breast cancer cells

Aberrant expression of Protein Arginine Methyltransferases (PRMTs) has been observed in several cancer types, including breast cancer. We previously reported that the PRMT1v2 isoform, which is generated through inclusion of alternative exon 2, is overexpressed in breast cancer cells and promotes their invasiveness. However, the precise mechanism by which expression of this isoform is controlled and how it is dysregulated in breast cancer remains unknown. Using a custom RNA interference-based screen, we identified several RNA binding proteins (RBP) which, when knocked down, altered the relative abundance of the alternatively spliced PRMT1v2 isoform. Amongst the top hits were SNW Domain containing 1 (SNW1) and RBP-associated with lethal yellow mutation (RALY), which both associated with the PRMT1 pre-mRNA and upon depletion caused an increase or decrease in the relative abundance of PRMT1v2 isoform mRNA and protein. Most importantly, a significant decrease in invasion was observed upon RALY knockdown in aggressive breast cancer cells, consistent with targeting PRMT1v2 directly, and this effect was rescued by the exogenous re-expression of PRMT1v2. We show that SNW1 expression is decreased, while RALY expression is increased in breast cancer cells and tumours, which correlates with decreased patient survival. This work revealed crucial insight into the mechanisms regulating the expression of the PRMT1 alternatively spliced isoform v2 and its dysregulation in breast cancer. It also provides proof-of-concept support for the development of therapeutic strategies where regulators of PRMT1 exon 2 alternative splicing are targeted as an approach to selectively reduce PRMT1v2 levels and metastasis in breast cancer.     

The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice

PRMT5 is a type II protein arginine methyltransferase with roles in stem cell biology, reprograming, cancer and neurogenesis. During embryogenesis in the mouse, it was hypothesized that PRMT5 functions with the master germline determinant BLIMP1 to promote primordial germ cell (PGC) specification. Using a Blimp1‐Cre germline conditional knockout, we discovered that Prmt5 has no major role in murine germline specification, or the first global epigenetic reprograming event involving depletion of cytosine methylation from DNA and histone H3 lysine 9 dimethylation from chromatin. Instead, we discovered that PRMT5 functions at the conclusion of PGC reprograming I to promote proliferation, survival and expression of the gonadal germline program as marked by MVH. We show that PRMT5 regulates gene expression by promoting methylation of the Sm spliceosomal proteins and significantly altering the spliced repertoire of RNAs in mammalian embryonic cells and primordial cells.     

A protein arginine N-methyltransferase 1 (PRMT1) and 2 heteromeric interaction increases PRMT1 enzymatic activity

Protein arginine N-methyltransferases (PRMTs) act in signaling pathways and gene expression by methylating arginine residues within target proteins. PRMT1 is responsible for most cellular arginine methylation activity and can work independently or in collaboration with other PRMTs. In this study, we demonstrate a direct interaction between PRMT1 and PRMT2 using co-immunoprecipitation, bimolecular fluorescence complementation, and enzymatic assays. As a result of this interaction, PRMT2 stimulated PRMT1 activity, affecting its apparent V(max) and K(M) values in vitro and increasing the production of methylarginines in cells. Active site mutations and regional deletions from PRMT1 and -2 were also investigated, which demonstrated that complex formation required full-length, active PRMT1. Although the inhibition of methylation by adenosine dialdehyde prevented the interaction between PRMT1 and -2, it did not prevent the interaction between PRMT1 and a truncation mutant of PRMT2 lacking its Src homology 3 (SH3) domain. This result suggests that the SH3 domain may mediate an interaction between PRMT1 and -2 in a methylation-dependent fashion. On the basis of our findings, we propose that PRMT1 serves as the major methyltransferase in cells by forming higher-order oligomers with itself, PRMT2, and possibly other PRMTs.     

Xenopus nonmuscle myosin heavy chain isoforms have different subcellular localizations and enzymatic activities [published erratum appears in J Cell Biol 1997 Jul 14;138(1):215]

There are two isoforms of the vertebrate nonmuscle myosin heavy chain, MHC-A and MHC-B, that are encoded by two separate genes. We compared the enzymatic activities as well as the subcellular localizations of these isoforms in Xenopus cells. MHC-A and MHC-B were purified from cells by immunoprecipitation with isoform-specific peptide antibodies followed by elution with their cognate peptides. Using an in vitro motility assay, we found that the velocity of movement of actin filaments by MHC-A was 3.3-fold faster than that by MHC-B. Likewise, the Vmax of the actin-activated Mg(2+)-ATPase activity of MHC-A was 2.6- fold greater than that of MHC-B. Immunofluorescence microscopy demonstrated distinct localizations for MHC-A and MHC-B. In interphase cells, MHC-B was present in the cell cortex and diffusely arranged in the cytoplasm. In highly polarized, rapidly migrating interphase cells, the lamellipodium was dramatically enriched for MHC-B suggesting a possible involvement of MHC-B based contractions in leading edge extension and/or retraction. In contrast, MHC-A was absent from the cell periphery and was arranged in a fibrillar staining pattern in the cytoplasm. The two myosin heavy chain isoforms also had distinct localizations throughout mitosis. During prophase, the MHC-B redistributed to the nuclear membrane, and then resumed its interphase localization by metaphase. MHC-A, while diffuse within the cytoplasm at all stages of mitosis, also localized to the mitotic spindle in two different cultured cell lines as well as in Xenopus blastomeres. During telophase both isoforms colocalized to the contractile ring. The different subcellular localizations of MHC-A and MHC-B, together with the data demonstrating that these myosins have markedly different enzymatic activities, strongly suggests that they have different functions.     

Targeting PRMT5/Akt signalling axis prevents human lung cancer cell growth

The emerging evidence reveals that protein arginine methyltransferase 5 (PRMT5) is involved in regulation of tumour cell proliferation and cancer development. Nevertheless, the exact role of PRMT5 in human lung cancer cell proliferation and the underlying molecular mechanism remains largely obscure. Here, we showed that PRMT5 was highly expressed in human lung cancer cells and lung cancer tissues. Furthermore, we generated PRMT5 stable knockdown cell lines (A549 and H1299 cells) and explored the functions of PRMT5 in lung cancer cell proliferation. We found that the down‐regulation of PRMT5 by shRNA or the inhibition of PRMT5 by specific inhibitor GSK591 dramatically suppressed cyclin E1 and cyclin D1 expression and cell proliferation. Moreover, we uncovered that PRMT5 promoted lung cancer cell proliferation via regulation of Akt activation. PRMT5 was directly co‐localized and interacted with Akt, but not PTEN and mTOR. Down‐regulation or inhibition of PRMT5 markedly reduced Akt phosphorylation at Thr308 and Ser473, whereas the expression of PTEN and mTOR phosphorylation was unchanged, indicating that PRMT5 was an important upstream regulator of Akt and induced lung cancer cell proliferation. Altogether, our results indicate that PRMT5 promotes human lung cancer cell proliferation through direct interaction with Akt and regulation of Akt activity. Our findings also suggest that targeting PRMT5 may have therapeutic potential for treatment of human lung cancer.     

A transient kinetic analysis of PRMT1 catalysis

Post-translational modifications (PTMs) are important strategies used by eukaryotic organisms to modulate their phenotypes. One of the well-studied PTMs, arginine methylation, is catalyzed by protein arginine methyltransferases (PRMTs) with SAM as the methyl donor. The functions of PRMTs have been broadly studied in different biological processes and diseased states, but the molecular basis for arginine methylation is not well-defined. In this study, we report the transient-state kinetic analysis of PRMT1 catalysis. The fast association and dissociation rates suggest that PRMT1 catalysis of histone H4 methylation follows a rapid equilibrium sequential kinetic mechanism. The data give direct evidence that the chemistry of methyl transfer is the major rate-limiting step and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, from the stopped-flow fluorescence measurements, we have identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. These results provide new insights into the mechanism of arginine methylation and the rational design of PRMT inhibitors.