A novel splicing isoform of protein arginine methyltransferase 1 (PRMT1) that lacks the dimerization arm and correlates with cellular malignancy.

Methylation of arginine residues is an important modulator of protein function that is involved in epigenetic gene regulation, DNA damage response and RNA maturation, as well as in cellular signaling. The enzymes that catalyze this post-translational modification are called protein arginine methyltransferases (PRMTs), of which PRMT1 is the predominant enzyme. Human PRMT1 has previously been shown to occur in seven splicing isoforms, which are differentially abundant in different tissues, and have distinct substrate specificity and intracellular localization. Here we characterize a novel splicing isoform which does not affect the amino-terminus of the protein like the seven known isoforms, but rather lacks exons 8 and 9 which encode the dimerization arm of the enzyme that is essential for enzymatic activity. Consequently, the isoform does not form catalytically active oligomers with the other endogenous PRMT1 isoforms. Photobleaching experiments reveal an immobile fraction of the enzyme in the nucleus, in accordance with earlier results from our laboratory that had shown a tight association of inhibited or inactivated PRMT1 with chromatin and the nuclear scaffold. Thus, it apparently is able to bind to the same substrates as catalytically active PRMT1. This isoform is found in a variety of cell lines, but is increased in those of cancer origin or after expression of the EMT-inducing transcriptional repressor Snail1. We discuss that the novel isoform could act as a modulator of PRMT1 activity in cancer cells by acting as a competitive inhibitor that shields substrates from access to active PRMT1 oligomers.

Senescence-associated microRNAs target cell cycle regulatory genes in normal human lung fibroblasts.

Senescence recapitulates the ageing process at the cell level. A senescent cell stops dividing and exits the cell cycle. MicroRNAs (miRNAs) acting as master regulators of transcription, have been implicated in senescence. In the current study we investigated and compared the expression of miRNAs in young versus senescent human fibroblasts (HDFs), and analysed the role of mRNAs expressed in replicative senescent HFL-1 HDFs. Cell cycle analysis confirmed that HDFs accumulated in G1/S cell cycle phase. Nanostring analysis of isolated miRNAs from young and senescent HFL-1 showed that a distinct set of 15 miRNAs were significantly up-regulated in senescent cells including hsa-let-7d-5p, hsa-let-7e-5p, hsa-miR-23a-3p, hsa-miR-34a-5p, hsa-miR-122-5p, hsa-miR-125a-3p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-503-5p, hsa-miR-574-3p, hsa-miR-574-5p and hsa-miR-4454. Importantly, pathway analysis of miRNA target genes down-regulated during replicative senescence in a public RNA-seq data set revealed a significant high number of genes regulating cell cycle progression, both G1/S and G2/M cell cycle phase transitions and telomere maintenance. The reduced expression of selected miRNA targets, upon replicative and oxidative-stress induced senescence, such as the cell cycle effectors E2F1, CcnE, Cdc6, CcnB1 and Cdc25C was verified at the protein and/or RNA levels. Induction of G1/S cell cycle phase arrest and down-regulation of cell cycle effectors correlated with the up-regulation of miR-221 upon both replicative and oxidative stress-induced senescence. Transient expression of miR-221/222 in HDFs promoted the accumulation of HDFs in G1/S cell cycle phase. We propose that miRNAs up-regulated during replicative senescence may act in concert to induce cell cycle phase arrest and telomere erosion, establishing a senescent phenotype.

Protein arginine methyltransferase 6 enhances polyglutamine-expanded androgen receptor function and toxicity in spinal and bulbar muscular atrophy.

Polyglutamine expansion in androgen receptor (AR) is responsible for spinobulbar muscular atrophy (SBMA) that leads to selective loss of lower motor neurons. Using SBMA as a model, we explored the relationship between protein structure/function and neurodegeneration in polyglutamine diseases. We show here that protein arginine methyltransferase 6 (PRMT6) is a specific co-activator of normal and mutant AR and that the interaction of PRMT6 with AR is significantly enhanced in the AR mutant. AR and PRMT6 interaction occurs through the PRMT6 steroid receptor interaction motif, LXXLL, and the AR activating function 2 surface. AR transactivation requires PRMT6 catalytic activity and involves methylation of arginine residues at Akt consensus site motifs, which is mutually exclusive with serine phosphorylation by Akt. The enhanced interaction of PRMT6 and mutant AR leads to neurodegeneration in cell and fly models of SBMA. These findings demonstrate a direct role of arginine methylation in polyglutamine disease pathogenesis.

Stable C0T-1 repeat RNA is abundant and is associated with euchromatic interphase chromosomes.

Recent studies recognize a vast diversity of noncoding RNAs with largely unknown functions, but few have examined interspersed repeat sequences, which constitute almost half our genome. RNA hybridization in situ using C0T-1 (highly repeated) DNA probes detects surprisingly abundant euchromatin-associated RNA comprised predominantly of repeat sequences (C0T-1 RNA), including LINE-1. C0T-1-hybridizing RNA strictly localizes to the interphase chromosome territory in cis and remains stably associated with the chromosome territory following prolonged transcriptional inhibition. The C0T-1 RNA territory resists mechanical disruption and fractionates with the nonchromatin scaffold but can be experimentally released. Loss of repeat-rich, stable nuclear RNAs from euchromatin corresponds to aberrant chromatin distribution and condensation. C0T-1 RNA has several properties similar to XIST chromosomal RNA but is excluded from chromatin condensed by XIST. These findings impact two "black boxes" of genome science: the poorly understood diversity of noncoding RNA and the unexplained abundance of repetitive elements.

Protein arginine methyltransferase 1 and 8 interact with FUS to modify its sub-cellular distribution and toxicity in vitro and in vivo.

Amyotrophic lateral sclerosis (ALS) is a late onset and progressive motor neuron disease. Mutations in the gene coding for fused in sarcoma/translocated in liposarcoma (FUS) are responsible for some cases of both familial and sporadic forms of ALS. The mechanism through which mutations of FUS result in motor neuron degeneration and loss is not known. FUS belongs to the family of TET proteins, which are regulated at the post-translational level by arginine methylation. Here, we investigated the impact of arginine methylation in the pathogenesis of FUS-related ALS. We found that wild type FUS (FUS-WT) specifically interacts with protein arginine methyltransferases 1 and 8 (PRMT1 and PRMT8) and undergoes asymmetric dimethylation in cultured cells. ALS-causing FUS mutants retained the ability to interact with both PRMT1 and PRMT8 and undergo asymmetric dimethylation similar to FUS-WT. Importantly, PRMT1 and PRMT8 localized to mutant FUS-positive inclusion bodies. Pharmacologic inhibition of PRMT1 and PRMT8 activity reduced both the nuclear and cytoplasmic accumulation of FUS-WT and ALS-associated FUS mutants in motor neuron-derived cells and in cells obtained from an ALS patient carrying the R518G mutation. Genetic ablation of the fly homologue of human PRMT1 (DART1) exacerbated the neurodegeneration induced by overexpression of FUS-WT and R521H FUS mutant in a Drosophila model of FUS-related ALS. These results support a role for arginine methylation in the pathogenesis of FUS-related ALS.

Human protein arginine methyltransferases in vivo--distinct properties of eight canonical members of the PRMT family.

Methylation of arginine residues is a widespread post-translational modification of proteins catalyzed by a small family of protein arginine methyltransferases (PRMTs). Functionally, the modification appears to regulate protein functions and interactions that affect gene regulation, signalling and subcellular localization of proteins and nucleic acids. All members have been, to different degrees, characterized individually and their implication in cellular processes has been inferred from characterizing substrates and interactions. Here, we report the first comprehensive comparison of all eight canonical members of the human PRMT family with respect to subcellular localization and dynamics in living cells. We show that the individual family members differ significantly in their properties, as well as in their substrate specificities, suggesting that they fulfil distinctive, non-redundant functions in vivo. In addition, certain PRMTs display different subcellular localization in different cell types, implicating cell- and tissue-specific mechanisms for regulating PRMT functions.

Nucleo-cytoplasmic shuttling of protein arginine methyltransferase 1 (PRMT1) requires enzymatic activity.

Methylation of arginine residues is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMT), of which PRMT1 is the predominant member in human cells. We have previously described the localization and mobility of PRMT1 in live cells, and found that it shuttles between the nucleus and the cytoplasm depending on the methylation status of substrate proteins. Recently, amino-terminal splicing isoforms of PRMT1 were shown to differ significantly in intracellular localization, the most interesting being splice variant 2 that carries a nuclear export signal in its amino terminus, and is expressed in increased levels in breast cancer cells. We show here that enzymatic activity is required for nucleo-cytoplasmic shuttling of PRMT1v2, as a catalytically inactive mutant highly accumulates in the nucleus and displays altered intranuclear mobility as determined by fluorescence recovery after photobleaching experiments. Our results indicate that nuclear export of PRMT1v2 is dominant over activity-independent nuclear import, but can only occur after activity-dependent release of the enzyme from substrates, suggesting that shuttling of the enzyme provides a dynamic mechanism for the regulation of substrate methylation.

Identifying functional neighborhoods within the cell nucleus: proximity analysis of early S-phase replicating chromatin domains to sites of transcription, RNA polymerase II, HP1gamma, matrin 3 and SAF-A.

Higher order chromatin organization in concert with epigenetic regulation is a key process that determines gene expression at the global level. The organization of dynamic chromatin domains and their associated protein factors is intertwined with nuclear function to create higher levels of functional zones within the cell nucleus. As a step towards elucidating the organization and dynamics of these functional zones, we have investigated the spatial proximities among a constellation of functionally related sites that are found within euchromatic regions of the cell nucleus including: HP1gamma, nascent transcript sites (TS), active DNA replicating sites in early S-phase (PCNA) and RNA polymerase II sites. We report close associations among these different sites with proximity values specific for each combination. Analysis of matrin 3 and SAF-A sites demonstrates that these nuclear matrix proteins are highly proximal with the functionally related sites as well as to each other and display closely aligned and overlapping regions following application of the minimal spanning tree (MST) algorithm to visualize higher order network-like patterns. Our findings suggest that multiple factors within the nuclear microenvironment collectively form higher order combinatorial arrays of function. We propose a model for the organization of these functional neighborhoods which takes into account the proximity values of the individual sites and their spatial organization within the nuclear architecture.

Local chromatin mobility is independent of transcriptional activity.

The dynamics of chromatin in live cells is characterized by high mobility on a sub-micrometer scale and strict constraints on larger scales. While there is ample evidence that chromatin loci can move within the nucleus in response to transcriptional activation, chromatin loci expressed at a steady state are confined to small intranuclear volumes, which they do not leave for hours. Previous work has shown, using in-vivo tagging of chromatin loci, that chromatin at the nuclear periphery has lower mobility and is more strictly constrained than chromatin in more internal nuclear positions. It appeared plausible that this difference was because of differences in local transcriptional activity and association of peripheral chromatin loci with heterochromatin. We here provide evidence that chromatin mobility is in fact independent of local transcriptional activity.

Protein arginine methyltransferases: guardians of the Arg?

The recent discovery of enzymes that convert methylated arginine residues in proteins to citrulline has catapulted arginine methylation into the attention of cell-signaling researchers. Long considered a rather static post-translational modification of marginal interest, it seems that arginine methylation has now joined the group of signaling pathways that operate via pairs of antagonistic enzymes. However, many questions remain unanswered, especially concerning the removal mechanism and its implication for the physiological role of arginine methylation. I propose that, in addition to the broadly discussed function as regulator of protein activity, arginine methylation might serve a second purpose: protection of arginine residues against attack by endogenous reactive dicarbonyl agents, such as methylglyoxal, which are natural by-products of normal metabolic pathways. Inefficient detoxification of these highly cytotoxic compounds results in inactivation of proteins that is causally linked to diabetes, cancer, neurodegenerative diseases and pathophysiologies of aging. This new concept of 'arginine protection' might have far-reaching implications for the development of drugs that exploit a natural protection mechanism for medical purposes.

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.

A stable proteinaceous structure in the territory of inactive X chromosomes.

Transcriptional inactivation of one copy of the X chromosome in female cells equalizes expression of X-linked genes between males and females. This "dosage compensation" is a multistep process that involves epigenetic modifications of chromatin and is induced by the expression of a large non-coding RNA termed Xist. In contrast to protein-coding mRNA molecules, which are free to diffuse and roam the entire nuclear interior, Xist is locally constrained to the territory of inactive X chromosomes by as yet unclear mechanisms. Recent results have suggested a contribution of scaffold attachment factor A (SAF-A) in the silencing of X-linked genes, maybe by inducing a local change in nuclear architecture. Here, in vivo mobility experiments demonstrate that SAF-A is a component of a highly stable proteinaceous structure in the territory of inactive X chromosomes, which might act as a platform for immobilizing Xist RNA during the maintenance phase of X inactivation.

Nuclear architecture and gene expression in the quest for novel therapeutics.

The architecture of the cell nucleus has long been a matter of debate, and is still not completely understood yet. However, much progress has been made in the last few years, gradually unraveling nuclear infrastructure and its importance of the regulation of key genetic events. It is now established that the readout of genetic information and its faithful duplication are not only affected by regulatory sequences in the genome, but also by their localization in the three-dimensional context and their relative position to functional subcompartments in the nucleus. Understanding how nuclear architecture and function are related and depend on each other has great potential to open up novel ways for the development of therapeutic agents. It is the purpose of this review to shed light on the role of nuclear architecture in regulating gene expression, and suggest that interfering with specific protein-protein interactions of transcription factors might provide new approaches to drug development.

Arginine methylation of scaffold attachment factor A by heterogeneous nuclear ribonucleoprotein particle-associated PRMT1.

Components of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex and other nucleic acid-binding proteins are subject to methylation on specific arginine residues by the catalytic activity of arginine methyltransferases. The methylation has been implicated in transcriptional regulation and RNA and protein trafficking and signal transduction, but the mechanism by which these functions are achieved has remained undetermined. We show here that the predominant arginine methyltransferase in human cells, protein arginine methyltransferase 1 (PRMT1), is associated with hnRNP complexes, dependent on the methylation status of the cell, and that it methylates its preferred substrates in situ. Binding of PRMT1 occurs through physical interaction with scaffold attachment factor A (SAF-A), also known as hnRNP-U, which is quantitatively methylated by PRMT1 in all investigated cell lines as determined by a novel, highly specific, methylation-sensitive antibody.

Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome.

The activation of mammalian origins of replication depends so far on ill understood epigenetic events, such as binding of transcription factors, chromatin structure, and nuclear localization. Understanding these mechanisms is not only a scientific challenge but also represents a prerequisite for the rational design of nonviral episomal vectors for mammalian cells. In this paper, we demonstrate that a tetramer of a 155-bp minimal nuclear scaffold/matrix attached region DNA module linked to an upstream transcription unit is sufficient for replication and mitotic stability of a mammalian episome in the absence of selection. Fluorescence in situ hybridization analyses, crosslinking with cis-diammineplatinum(II)-dichloride and chromatin immunoprecipitation demonstrate that this vector associates with the nuclear matrix or scaffold in vivo by means of specific interaction of the nuclear scaffold/matrix attached region with the nuclear matrix protein SAF-A. Results presented in this paper define the minimal requirements of an episomal vector for mammalian cells on the molecular level.

Localization and dynamics of small circular DNA in live mammalian nuclei.

While genomic DNA, packaged into chromatin, is known to be locally constrained but highly dynamic in the nuclei of living cells, little is known about the localization and dynamics of small circular DNA molecules that invade cells by virus infection, application of gene therapy vectors or experimental transfection. To address this point, we have created traceable model substrates by direct labeling of plasmid DNA with fluorescent peptide nucleic acids, and have investigated their fate after microinjection into living cells. Here, we report that foreign DNA rapidly undergoes interactions with intranuclear structural sites that strongly reduce its mobility and restrict the DNA to regions excluding nucleoli and nuclear bodies such as PML bodies. The labeled plasmids partially co-localize with SAF-A, a well characterized marker protein for the nuclear 'scaffold' or 'matrix', and are resistant towards extraction by detergent and, in part, elevated salt concentrations. We show that the localization and the low mobility of plasmids is independent of the plasmid sequence, and does not require the presence of either a scaffold attachment region (SAR) DNA element or a functional promoter.

Scaffold attachment factor A (SAF-A) is concentrated in inactive X chromosome territories through its RGG domain.

Female mammalian cells inactivate transcription from one of their X chromosomes to equalize gene expression of X-linked genes between males and females. Inactivation is a multistep process that involves a large non-coding RNA termed XIST, a variety of epigenetic modifications of chromatin, and alterations in protein composition such as enrichment of the histone variant macroH2A. We show here that inactive X chromosomes are also enriched in a well-characterized protein component of the nuclear scaffold, SAF-A. This protein has been implicated in chromatin organization, owing to its high specificity for scaffold-associated region (SAR)-DNA, in transcriptional regulation, e.g. of hormone-regulated genes, owing to its functional interaction with steroid receptors, and in RNA processing, owing to its interaction with RNA and heterogeneous nuclear ribonucleoprotein (hnRNP) particles. After near complete removal of DNA and associated chromatin proteins such as macroH2A, SAF-A remains with the "nuclear matrix", still highlighting the former position of inactive X chromosomes. Interestingly, the enrichment of SAF-A in the inactive X chromosome depends on the RNA binding domain of the protein, the RGG box, raising the possibility that interaction of SAF-A with XIST RNA may contribute to the silencing of X-linked genes by local changes in nuclear architecture.

An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo.

pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.