Versatility of PRMT5-Induced Methylation in Growth Control And

TIBS-871; No. of Pages 9

Review

Versatility of PRMT5-induced

methylation in growth control and development

1 2 3 2

Vrajesh Karkhanis , Yu-Jie Hu , Robert A. Baiocchi , Anthony N. Imbalzano and 1

Saı¨d Sif

Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, OH 43210, USA

Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA

Division of Hematology–Oncology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus,

OH 43210, USA

Arginine methylation governs important cellular pro- methyltransferases (PRMTs) specializes in arginine meth-

cesses that impact growth and proliferation, as well ylation of both histones and other cellular proteins. PRMT

as differentiation and development. Through their ability family members play a pivotal role in the regulation of

to catalyze symmetric or asymmetric methylation of diverse cellular processes that range from transcription

histone and non-histone proteins, members of the pro- and RNA processing to signaling, differentiation, apoptosis

tein arginine methyltransferase (PRMT) family regulate and tumorigenesis [4,5]. PRMT enzymes are classiﬁed into

chromatin structure and expression of a wide spectrum two subgroups depending on the type of modiﬁcation they

of target genes. Unlike other PRMTs, PRMT5 works in catalyze. Type I PRMT enzymes (PRMT1–4, PRMT6 and

G G G

concert with a variety of cellular proteins including ATP- PRMT8) generate v-N -monomethyl and v-N ,N -asym-

dependent chromatin remodelers and co-repressors to metric dimethyl arginines, whereas type II PRMT enzymes

induce epigenetic silencing. Recent work also implicates (PRMT5, PRMT7 and PRMT9) catalyze the formation of v-

G G 0G

PRMT5 in the control of growth-promoting and pro- N -monomethyl and v-N ,N -symmetric dimethyl argi-

survival pathways, which demonstrates its versatility nine residues [6]. Previous reviews have summarized what

as an enzyme involved in both epigenetic regulation is known about most PRMTs [4,5]; however, a signiﬁcant

of anti-cancer target genes and organelle biogenesis. amount of work has recently been published that describes

These studies not only provide insight into the molecular the role of PRMT5 in a variety of cellular processes includ-

mechanisms by which PRMT5 contributes to growth ing ribosome biogenesis [7], assembly of the Golgi appara-

control, but also justify therapeutic targeting of PRMT5. tus [8], cellular differentiation [9–11] and germ cell

speciﬁcation [12,13]. The relevance of PRMT5 in cancer

PRMT5-mediated arginine methylation extends beyond biology has also become evident, because more studies

epigenetics have shown that PRMT5 is involved in the regulation of

Modulation of chromatin structure involves many chroma- major signaling pathways that impact cell death and ma-

tin-remodeling factors, including enzymes that can post- lignant transformation [14–24]. In this review, we high-

translationally modify histones, and it is well established light some of the recent work that further characterizes the

that histone modiﬁcation represents a fundamental step in biological function of PRMT5 and that implicates it in the

the regulation of DNA accessibility during various cellular control of cell proliferation and development.

processes such as transcription, replication and DNA repair

[1,2]. Conversion of chromatin from a repressed form to a PRMT5 modulates cell growth and transformation

more open and active state and vice versa relies on coordi- PRMT5 is a type II methyltransferase that is associated

nated interplay between histone-modifying enzymes, ATP- with multiple protein complexes including human SWI/

dependent chromatin remodelers and DNA-modifying SNF chromatin remodelers (Figure 1a) [25]. As part of the

enzymes. The importance of chromatin modiﬁcation by SWI/SNF complex, PRMT5 hypermethylates promoter

these enzymes is reﬂected in their critical roles in control histones H3R8 and H4R3, and triggers transcriptional

of gene expression, cell proliferation and organismal devel- silencing of cell cycle regulatory and tumor suppressor

opment. genes (Figure 1b) [25–27]. Knockdown of PRMT5 expres-

Histones undergo a variety of post-translational sion is associated with slow growth, whereas PRMT5 over-

modiﬁcations in their globular domains and N-terminal expression causes cellular hyperproliferation. PRMT5

tails. Among the known histone modiﬁcations, lysine acet- levels are elevated in a variety of transformed cells, and

ylation and methylation have been the most studied [3]. it seems that this is a direct result of increased translation

A distinct group of enzymes termed protein arginine [26,27]. miRNA proﬁling in chronic lymphocytic leukemia,

mantle cell lymphoma cell lines and clinical samples has

Corresponding author: Sif, S. ([email protected]). revealed that aberrant PRMT5 expression is inversely

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(a) SWI/SNF mSIN3A/HDAC NF-E4 cyclin D1 T268A CDK4

BLIMP1

MBD2-NURD COPR5 PRMT5 SKI

HDAC3 AJUBA/SNAIL SMAD 2/3/4 N-CoR/SMRT

PDCD4 STRAP CEP-1/p53 p53 CBP JMY CBP

(b) Transcriptional silencing

me me me me - ST7, RBL2, SMAD7 Enhanced cell growth and proliferation Chromatin remodelers H3R8 H4R3 (SWI/SNF, NuRD) - CUL4A/B Altered DNA replication and genomic instability

Co-repressors - CDKN2A, cyclin E (CCNE) Misregulation of cell cycle progression (N-CoR/SMRT, AJUBA,COPR5) Or PRMT5 - egl-1 Stimulation of germ cell survival

Transcription factors, co-activators me me (p53,CBP) - E-cadherin (CDH1) Enhanced cell invasion and metastasis H4R3 me

CpG Developmental regulators - DHX38 Inhibition of germ cell specification (BLIMP1, NF-E4/DNMT3A) - γ -globin Silencing of developmentally regulated genes

TiBS

Figure 1. PRMT5-induced epigenetic and post-translational changes have a significant impact on cell growth and proliferation. Through multiple interactions with specific

transcription factors and chromatin-modifying enzymes, PRMT5 is capable of controlling critical cellular pathways. (a) PRMT5 associates with specific ATP-dependent

chromatin remodelers, co-repressors and co-activators, and controls expression of key target genes involved in growth control, metastasis, differentiation and

development. (b) PRMT5 alone or in combination with MEP50 interacts with a variety of transcriptional regulators to epigenetically modify specific histone arginine

residues, including H3R8 and H4R3, and to induce gene silencing. PRMT5-induced H4R3 methylation orchestrates recruitment of repressor DNA methyltransferase

DNMT3A. PRMT5 also promotes transcriptional silencing by post-translationally modifying chromatin-binding proteins such as MBD2, p53 and CBP, and altering their

biochemical properties.

correlated with levels of PRMT5-speciﬁc miRNAs. Over- (CDK4) to repress CUL4A/B transcription and thereby

expression of any of these miRNAs restored PRMT5 to its stabilize CDT1 (Figure 1a,b) [16]. As cells traverse the G1

basal levels, which supports the notion that they are to S phase, cyclin D1 is phosphorylated on T286 and is

directly involved in translational regulation of PRMT5. exported to the cytoplasm, where it undergoes ubiquitin-

Elevated expression of PRMT5 in transformed B cells dependent proteolysis [15]; substitutions of T286 render the

correlates with hypermethylation of H3R8 and H4R3 protein resistant to phosphorylation and subsequent degra-

[26,27], and global gene proﬁling combined with chromatin dation. The consequence is increased cyclin D1–CDK4 ac-

immunoprecipitation (ChIP) studies revealed that SWI/ tivity, enhanced CUL4A/B repression and stabilization of

SNF-associated PRMT5 negatively controls transcription CDT1, which leads to DNA re-replication and transforma-

of growth inhibitory genes [26,27]. The fact that PRMT5 is tion [16]. These conclusions were supported by afﬁnity

overexpressed in cancer cells and that it epigenetically puriﬁcation of ﬂag-tagged cyclin D1 T286A, which associat-

silences speciﬁc tumor suppressors shows that PRMT5 ed with PRMT5, its binding partner, methylosome protein

controls the growth of transformed B cells, and suggests 50 (MEP50) and the BRG1 SWI/SNF ATPase during the G1

that therapeutic targeting of PRMT5 might help to restore to S transition. Blocking of PRMT5/MEP50 methyltransfer-

expression of anti-cancer genes and inhibit growth of ase activity by knocking down PRMT5 expression or by

cancerous cells. using global PRMT inhibitors resulted in CUL4A/B tran-

PRMT5 also participates in transcriptional silencing of scriptional derepression and CDT1 degradation during S

the cullins CUL4A and CUL4B, which encode scaffolding phase, which indicates that PRMT5 is involved in the

proteins for the E3 ligase that targets chromatin licensing epigenetic silencing of key target genes that regulate levels

and DNA replication factor 1 (CDT1) for degradation during of the replication-licensing factor CDT1 [16].

S phase. Recent work demonstrated that PRMT5 mediates Regulation of cell-cycle control genes can also be medi-

the ability of mutant cyclin D1–cyclin-dependent kinase 4 ated by association of PRMT5 with other ATP-dependent

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chromatin remodelers. Afﬁnity puriﬁcation of NuRD remo- of developing cancer [34–37]. Additional alterations to this

deling enzymes showed that NuRD complexes containing residue have also been found in other tumors, which high-

the methyl-CpG binding domain protein 2 (MBD2) subunit lights the importance of arginine methylation in the con-

also contain PRMT5 and its binding partner MEP50 trol of p53-mediated events [38].

(Figure 1a) [28]. The presence of PRMT5/MEP50 in A more recent study in Caenorhabditis elegans showed

MBD2–NuRD requires the MBD2 N-terminal region, that PRMT-5 negatively regulates DNA damage-induced

which contains 14 RG repeats and is methylated by apoptosis, as evidenced by an increase in germ cell death

PRMT5. ChIP studies showed that PRMT5 co-localized on gamma-ray irradiation in worms lacking PRMT-5 [21].

with MBD2 and other NuRD subunits on the promoters When PRMT-5 was reintroduced, there was a clear de-

of several target genes, including CDKN2A (which encodes crease in germ cell apoptosis, which indicates that PRMT-5

ARF INK4a

p14 and p16 ) (Figure 1b). Furthermore, enrich- antagonizes DNA-damage-induced apoptosis in C. elegans.

ment of PRMT5–MBD2–NuRD depends on DNA methyla- Genetic inactivation of components of the core cell death

tion, because treatment with the hypomethylating agent 5- pathway indicated that the mechanism by which PRMT-5

aza-2 -deoxycytidine (5-azadC) reduced its association inhibits DNA-damage-induced apoptosis is through tran-

with target promoters [28]. In a separate study, the scriptional suppression of the CEP-1 (the p53 ortholog)

PRMT5–NuRD interaction was conﬁrmed, and PRMT5 target gene egl-1, an early driver of cell death (Figure 1b).

knockdown had a positive effect on the interaction between Association of PRMT-5 with CEP-1 and the co-activator

MBD2 and histone deacetylase (HDAC) complexes. More- CBP-1 resulted in methylation of CBP-1 R234, a site also

over, biochemical characterization of PRMT5–methylated conserved in human p300 and CBP. Methylation of CBP-1

MBD2–NuRD indicated that it had lower afﬁnity for meth- inhibited its ability to acetylate CEP-1/p53 and probably

ylated DNA. These studies suggest that methylation of histones, which thus caused transcriptional repression of

MBD2 has an inhibitory effect on its interaction with egl-1 and promoted germ cell survival [21]. Collectively,

HDAC repressor complexes and alters its binding proper- these ﬁndings demonstrate that PRMT-5 can target p53-

ties to the methylated DNA, thereby triggering derepres- regulated pathways by directly modifying p53 and altering

sion of MBD2–NuRD target genes [29]. its biochemical properties or by post-translationally modi-

A more recent report showed that the proto-oncogene fying its associated co-activators and affecting their cata-

SKI, which encodes a nuclear protein that functions lytic activity.

through interaction with SMAD2/3/4, N-CoR/SMRT and Further support for a role of PRMT5 in cell cycle control

HDAC3, also interacts with PRMT5 (Figure 1a). Biochem- and p53 tumor suppressor function came from work dem-

ical characterization of the SKI complex revealed that it onstrating that reduced PRMT5 expression inhibits MCF-

can deacetylate and methylate histones in vitro, and re- 7 breast cancer cell proliferation and the transition from

cruitment studies demonstrated that the SKI complex G1 to S. Treatment of MCF-7 cells with doxorubicin en-

binds the promoter region of the growth inhibitor target hanced the stability of p53 and increased expression of p53

gene SMAD7 (Figure 1b). Moreover, siRNA-mediated target genes, including MDM2 and CDKN1A (which

knockdown studies showed that PRMT5, HDAC3 and encodes p21). However, PRMT5 knockdown resulted in

SKI mediate transcriptional repression of SMAD7 in the decreased p53 stability and target gene expression, and

absence of transforming growth factor (TGF)-b. These ChIP assays indicated that decreased expression of

studies demonstrate that PRMT5 can act in concert with CDKN1A was caused by reduced p53 recruitment to its

other histone-modifying enzymes to regulate expression of promoter, which indicates that PRMT5 is required for

target genes that control cell growth and proliferation [17]. multiple aspects of p53 function in response to genotoxic

Among non-histone proteins targeted by PRMT5 is the stress [18].

p53 tumor suppressor protein, which is involved in the The concept of functional interactions between PRMT5

regulation of cell proliferation, cell cycle progression and and known tumor suppressors is also demonstrated by

cell death [18]. Under normal conditions, p53 protein work showing that programmed cell death protein 4

expression is maintained at low levels via E3 ubiquitin (PDCD4), a prognostic indicator for several tumor types

ligase MDM2-mediated proteolysis. On DNA damage, p53 [39] and a regulator of translation [40], associates with and

is activated and dissociates from MDM2, and its levels is methylated by PRMT5. In an orthotopic breast tumor

increase due to enhanced protein stability [30]. Numerous model, overexpression of both PRMT5 and PDCD4 en-

post-translational modiﬁcations to p53 that either enhance hanced tumor growth in a manner dependent on both

or inhibit its tumor suppressor activity have also been PRMT5 catalytic activity and the PDCD4 residue targeted

reported [31–33]. In particular, PRMT5 interacts with by PRMT5. Examination of patient samples showed a

and methylates p53 at R333, R335 and R337 on DNA correlation between elevated PDCD4 and PRMT5 expres-

damage (Figure 1a). The consequence is altered recruit- sion and poor outcome [41], which reﬂects the relevance of

ment to target genes in a promoter-speciﬁc manner and PRMT5 level and function in patient survival.

inhibition of p53 oligomerization (Figure 1b) [19]. Although Another important pathway regulated by PRMT5

their occurrence is rare, mutations that alter p53 R333 and involves the SNAIL transcription factor, which is involved

R337 have been detected; indeed, substitution of p53 in the regulation of embryonic development and metasta-

R337 to either a histidine or cysteine is known to destabi- sis. SNAIL regulates epithelial to mesenchymal transition

lize p53 and is associated with human cancer. Moreover, by altering cell adhesion through transcriptional repres-

p53 R337C substitution occurs in individuals with Li sion of the E-cadherin gene (CDH1). Several transcription-

Fraumeni syndrome, a disorder known to increase the risk al co-repressors interact with SNAIL, including AJUBA,

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which can bridge multiple interactions with several chro- that PRMT5 is crucial for germ cell formation. Loss of dart5

matin-modifying enzymes. Recent work showed that (also called Capsuleen), the ﬂy ortholog of PRMT5, causes a

AJUBA forms a complex with PRMT5 and recruits it to defect in spermatocyte maturation and infertility. By con-

SNAIL-repressed CDH1 (Figure 1a,b) [20]. Knockdown of trast, female dart5 mutants are fertile; however, their

either AJUBA or PRMT5 abrogated their association with embryos fail to form the pole cells that give rise to germ

the CDH1 promoter and resulted in transcriptional dere- cells in adults [44]. Dart5 enzyme activity is required for

pression of CDH1. These studies broaden our view of the methylation of spliceosomal proteins in oocytes and con-

impact of PRMT5 on cell growth and tumorigenesis by trols the localization of proteins in the pole plasm that are

indicating a role for PRMT5 in cell adhesion. essential for germ cell formation [45]. These ﬁndings sug-

Association of PRMT5 with various binding partners gest that PRMT5 might regulate germ cell formation by

seems to inﬂuence its substrate speciﬁcity. A yeast two- epigenetic and non-epigenetic mechanisms.

hybrid assay identiﬁed a nuclear protein dubbed coopera- PRMT5 can also promote somatic cell reprogramming

tor of PRMT5 (COPR5) as a PRMT5-interacting partner [46], consistent with ﬁndings from studies of early devel-

(Figure 1a). COPR5-associated PRMT5 preferentially opment, ES cells and germ cells. Knockdown of PRMT5

methylates histone H4R3 in vitro; however, when PRMT5 reduced the reprogramming efﬁciency with classical

is puriﬁed from cells in which COPR5 is knocked down, Yamanaka factors (OCT3/4, SOX2, KLF4, MYC); however,

methylation of histone H3 is more prevalent, which sug- the mechanism remains undetermined. Intriguingly, a

gests that the COPR5–PRMT5 complex methylates H4 type I PRMT inhibitor that enabled OCT4-induced mouse

more efﬁciently than H3. In vivo both COPR5 and PRMT5 embryonic ﬁbroblast (MEF) reprogramming in combina-

are enriched at the cyclin E (CCNE) promoter, where the tion with a TGF-b inhibitor was recently identiﬁed [47],

PRMT5-induced epigenetic mark H4R3 is hypermethy- which suggests that type I PRMTs might negatively regu-

lated; however, COPR5 knockdown leads to transcriptional late the reprogramming process. Thus, it seems that

derepression of CCNE (Figure 1b) [42]. These experiments PRMT5 and one or more type I PRMTs might have oppos-

show that COPR5 is required for PRMT5 recruitment to ing functions in somatic cell reprogramming.

the CCNE promoter and indicate that COPR5 directs Tissue-speciﬁc gene expression is also regulated by

PRMT5 methyltransferase activity towards H4R3. PRMT5. Using the human b-globin locus as a model, a

link has been established between PRMT5-mediated his-

PRMT5 functions as a co-repressor as well as a co- tone methylation, DNA methylation and gene repression.

activator during development and differentiation Expression of globin genes undergoes developmental

A role for PRMT5 during embryonic development was re- stage-speciﬁc regulation during which expression of the

cently demonstrated through studies that showed that fetal speciﬁc g-globin gene precedes that of the adult

Prmt5-null mice suffer from early embryonic lethality be- speciﬁc b-globin gene. In adult bone-marrow erythroid

tween embryonic days 3.5 and 6.5 [43]. In addition, the cells, transcriptional silencing of the genes encoding fetal

failure to derive embryonic stem (ES) cells from Prmt5-null (g) globin is mediated by the transcriptional factor NF-E4,

blastocysts and the downregulation of pluripotency genes as which can interact with PRMT5 (Figure 1a) [48]. Recruit-

differentiation genes were upregulated in PRMT5 knock- ment of PRMT5 results in H4R3 methylation, which in

down ES cells strongly suggest that PRMT5 is required for turn serves as a direct binding site for DNA methyltrans-

stem cell pluripotency. Biochemical analysis indicated that ferase DNMT3A and subsequent DNA methylation of CpG

PRMT5 methylated histone H2A before its incorporation dinucleotides ﬂanking the transcriptional start site of the

into chromatin, an event that correlated with repression of g-globin gene (Figure 1b) [49]. PRMT5 binding and enzyme

differentiation genes. Furthermore, the physical interaction activity are crucial for the recruitment of a repressive

between PRMT5 and signal transducer and activator of multisubunit complex containing DNMT3A, HDAC1,

transcription (STAT)3 suggested that PRMT5 might also SUV4-20h1 and casein kinase (CK)2a to the g-globin pro-

repress differentiation genes in ES cells through the leuke- moter. Remarkably, PRMT5 also inﬂuenced other epige-

mia inhibitory factor (LIF)–STAT3 signaling pathway. netic marks including H4S1 ph, H4K20me3, and to some

PRMT5 has also been implicated in epigenetic regula- extent H3K9me3 and H3K27me3, which were reduced in

tion during mouse germ cell speciﬁcation by directly inter- cells in which PRMT5 function was abrogated [49,50].

acting with the transcriptional repressor B-lymphocyte- These experiments show that recruitment of PRMT5 is

induced maturation protein (BLIMP)1 (Figure 1a) [12]. crucial for coordinated binding of repressive chromatin-

Translocation of the BLIMP1–PRMT5 complex from the modifying enzymes to the developmentally regulated g-

nucleus to the cytoplasm during embryogenesis results in globin gene.In contrast to the roles of PRMT5 in develop-

H2AR3/H4R3 hypomethylation and derepression of genes mental programs discussed thus far, analysis of skeletal

involved in germ cell speciﬁcation (Figure 1b). Moreover, muscle differentiation indicates that PRMT5 acts as a

BLIMP1 expression in embryonic P19 carcinoma cells transcriptional co-activator. Knockdown of PRMT5 inhib-

results in repression of germ cell speciﬁcation genes and ited myogenesis in culture, and ChIP analysis indicated

hypermethylation of histones H2AR3 and H4R3 at these that PRMT5 binding and PRMT5-mediated H3R8 methyl-

loci. A role for BLIMP1, PRMT5 and H2A/H4R3 dimethy- ation inducibly occurred at myogenic promoters shortly

lation in human fetal germ cell development has also been after the onset of differentiation [9,10]. Analysis of the

demonstrated, which highlights the relevance of this mech- myogenin promoter revealed that the absence of PRMT5

anism in maintenance of the undifferentiated and pluripo- prevented binding and chromatin remodeling by the SWI/

tent state [12,13,43]. Studies in Drosophila also revealed SNF enzyme, an obligatory step for myogenin expression,

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which indicates that the co-regulatory function of PRMT5 potentiates TRAIL-induced tumor cell death, which indi-

and SWI/SNF is context-dependent. Interestingly, this cates that PRMT5 inhibits the killing effect of TRAIL [24].

work indicated a temporal requirement for PRMT5 in The molecular mechanism is independent of the PRMT5

differentiation, given that gene activation events subse- methyltransferase activity and involves nuclear factor

quent to myogenin expression did not directly require (NF)-kB, because TRAIL-induced IKK activation and

PRMT5. Instead, a type I PRMT, PRMT4, was required IkB degradation were reduced, as was expression of NF-

for these gene activation events [10,11]. Unlike the case of kB target genes, in PRMT5 knockdown cells. PRMT5

somatic cell reprogramming, in which the different PRMTs knockdown sensitizes tumoral, but not normal, cells to

might work in opposition, these data indicate apparently TRAIL-induced death, and PRMT5 overexpression endows

independent roles for PRMT4 and PRMT5 in the same a cancer cell line with increased resistance to TRAIL-

biological process (PRMT5 is required for early myogenic induced death, which suggests that targeting of PRMT5

gene activation, whereas PRMT4 is not; by contrast, in TRAIL-based therapies might increase the efﬁciency of

PRMT5 is not required for late myogenic gene expression tumor cell targeting [24].

whereas PRMT4 is). Cross-talk between PRMT5 and signaling molecules has

been demonstrated, and oncogenic mutations in PRMT5-

PRMT5 methylates multiple targets in the cytoplasm interacting kinases have been implicated in tumorigenesis.

and thus modulates growth-promoting and cell death- The PRMT5–MEP50 complex can methylate members of

inducing pathways the Janus kinase (JAK) family (Figure 2a). Although the

The cellular localization of PRMT5 differs between non- physical interaction between PRMT5 and JAK2 was iden-

transformed and transformed cells. In most primary and tiﬁed more than a decade ago [54], the functional signiﬁ-

immortalized cells, PRMT5 is primarily located in the cance of this interaction was only recently unraveled. JAK2

cytosol, with a small amount in the nucleus; however, this is a tyrosine kinase that binds to the cytoplasmic domain of

distribution is reversed in transformed cells [25,27]. There- hematopoietic cytokine receptors, which are activated by

fore, it seems that PRMT5 localization and the substrates ligands such as erythropoietin and granulocyte colony

it targets in each cellular compartment might play a stimulating factor, and transduces signaling by phosphor-

crucial role in the control of cell growth and proliferation. ylating downstream targets (Figure 2b). Activation or

Epidermal growth factor receptor (EGFR) is a transmem- oncogenic mutation impairs negative regulation of JAK2

brane receptor that undergoes several post-translational and results in constitutive activation of its tyrosine kinase

modiﬁcations that inﬂuence its biological activity. Activa- activity, which in turn enhances downstream signaling

tion of EGFR induces its kinase activity and autophosphor- cascades and promotes cellular proliferation [55]. JAK2

ylation, which in turn initiates downstream signaling mutations have been detected in the majority of patients

events associated with cellular proliferation and tumori- with myeloproliferative neoplasms, a type of stem cell

genesis. EGFR interacts and co-localizes with PRMT5 disorder characterized by enhanced proliferation of he-

(Figure 2a) and PRMT5 knockdown reduces monomethy- matopoietic stem cells [56–58]. Interestingly, constitutive-

lation of EGFR R1175, which suggests that PRMT5 is ly active JAK2 V617F and K539L mutant kinases bound

directly involved in EGFR methylation. Furthermore, com- more avidly to and phosphorylated PRMT5 both in vitro

parison of breast cancer cell lines expressing either wild- and in vivo, and impaired its methyltransferase activity by

type or mutant EGFR R1175K indicated that EGFR meth- disrupting its interaction with MEP50 and affecting global

ylation suppressed its biological function, as demonstrated H2AR3 and H4R3 methylation [23]. By contrast, wild-type

by increased growth, efﬁcient tumor formation and greater JAK2, even when activated, did not phosphorylate

migration and invasion capabilities of cells expressing PRMT5. These studies demonstrate that oncogenic

mutant EGFR (Figure 2b). Methylation of R1175 down- JAK2 kinases promote myeloproliferative disease and ery-

regulates EGFR function by promoting phosphorylation of throid differentiation, at least in part, by inactivating

Tyr1173, a binding site for SHP1, a protein tyrosine phos- PRMT5, and suggest that in the context of hematopoietic

phatase that inhibits ERK signaling. Thus, it is conceiv- cells, inhibition of PRMT5 activity might impinge on EGFR

able that PRMT5-mediated EGFR methylation leads to signaling, thereby inducing cell proliferation and tumori-

reduced ERK signaling, whereas demethylation or substi- genesis.

tution of R1175 could have the opposite effect. It is also PRMT5–MEP50-mediated arginine methylation has

possible that in transformed cells, relocalization of PRMT5 been implicated in a variety of cytosolic processes, includ-

from the cytosol to the nucleus could result in reduced ing biogenesis of Sm-class ribonucleoproteins. Sm proteins

EGFR methylation and potentiate ERK signaling, which in are components of the spliceosomal U1, U2, U4 and U5

turn could enhance the growth of cancer cells [22]. small nuclear ribonucleoproteins (snRNPs). PRMT5, as

PRMT5 can also antagonize pro-apoptotic signaling well as PRMT7, another type II PRMT, methylates three

pathways. The tumor necrosis factor (TNF)-related apo- of the seven Sm proteins and enhances their binding to the

ptosis-inducing ligand (TRAIL) is a member of the TNF survival motor neuron (SMN) complex (Figure 2a,b) [59].

superfamily that has anti-tumor activity and immense Interaction of methylated Sm proteins with the SMN

therapeutic potential [51]. Binding of TRAIL to death complex facilitates their loading onto the Sm site of small

receptors (DR4 and DR5) assembles the death-inducing nuclear RNA (snRNA), which thereby gives rise to U-

signaling complex (DISC), which induces apoptosis [52,53]. snRNPs. Knockdown of either PRMT5 or PRMT7 reduces

Recent work showed that PRMT5 physically interacts with symmetric methylation of Sm proteins and disrupts

DR4 and DR5 (Figure 2a,b) and that knockdown of PRMT5 snRNP assembly. In a separate investigation designed

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(a) EGFR PIWI proteins (mili/miwi/miwi2) DR4, DR5

PRMT5 JAK RPS10 pICIn

RioK

SmD1, SmD3, SmB/B’ GM130

Nucleolin

(b) EPO TRAIL EGF D D R R E E PRMT5 PRMT5 receptor

4 5 Cytokine PRMT5 G G me JAK F F R1175 PRMT5 R R me MEP50 R1175K Y1173 Downstream Mutant P JAK Ikk Assembly of signaling DISC complex SHP1 Activation P ERK signaling Growth control PRMT5 NF-κB Apoptosis MEP50 Activation Cellular proliferation and Hypomethylation of tumorigenesis H2AR3 and H4R3 Induction of pro-survival genes Hyperproliferation of Hematopoietic stem cells me me PRMT5 SmB/B’ me me me me GM130 SmD3 SmE me me me RPS10 SmF me SmD2 40S SmD1 SmG 60S

Enhanced binding to SMN complex Ribosome assembly Stabilization of golgi and spliceosomal U-SnRNP

TiBS

Figure 2. Role of PRMT5 in cell signaling and organelle biogenesis. Association of PRMT5 with various receptors, signaling molecules and organelle subunits enables it to

impact cell proliferation, tumorigenesis and cell death. (a) PRMT5 targets multiple proteins in the cytoplasm and modulates signaling pathways, RNA processing and

organelle assembly. (b) Methylation of EGFR by PRMT5 has an inhibitory effect on ERK signaling and affects cellular proliferation, migration and invasion, whereas

interaction of PRMT5 with death receptors DR4 and DR5 leads to inhibition of TRAIL-mediated apoptosis and induction of pro-survival gene expression. In the context of

pluripotent stem cells, phosphorylation of PRMT5 by oncogenic JAK kinase mutants results in loss of its methyltransferase activity and hyperproliferation of hematopoietic

stem cells. Through its ability to interact with and methylate various cytoplasmic proteins, PRMT5 is also involved in assembly of spliceosomal proteins, Golgi apparatus

and the 40S ribosomal subunit.

to discover proteins affected by lack of methylation, two provided conﬁrmation of the role played by PRMT5 in

Arabidopsis thaliana glycine-rich RNA-binding proteins RNA processing [60]. In addition to its association with

(AtGRP7 and 8), which are homologs of the human hetero- MEP50, PRMT5 also forms a complex with either pICln

geneous nuclear ribonucleoprotein (hnRNP) A1 and (chloride channel, nucleotide sensitive 1A), which recruits

hnRNP A2/B1 that are known to inhibit pre-mRNA splic- Sm proteins, or the Rio-domain-containing protein kinase

ing, were identiﬁed as PRMT5 substrates, which thus (RIOK)1, which competes with pICln for PRMT5 binding

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(Figure 2a) [61,62]. Just as interaction of PRMT5 with were shown to be PRMT5 substrates, and alterations to

COPR5 or BLIMP1 restricts its methyltransferase activity speciﬁc arginine residues within the N-terminal region of

to speciﬁc histones, association of PRMT5 with either the Piwi protein MILI resulted in loss of interaction and

pICln or RIOK1 directs its catalytic activity toward Sm colocalization with Tudor proteins, which suggests that

proteins or nucleolin, respectively. In addition to its in- PRMT5-induced methylation of Piwi proteins is crucial for

volvement in regulating rDNA transcription and pre-ribo- their recognition by the Tudor family of proteins and

some packaging, nucleolin also functions as a shuttle proper function of the piRNA pathway [64].

protein that transports viral and cytoplasmic proteins

between the cytoplasm and nucleus. The relevance of Concluding remarks

the PRMT5–nucleolin complex became apparent when it Biochemical and physiological characterization has dem-

was shown that the cancer cell growth inhibitor AS1411, a onstrated that PRMT5 is crucial for maintenance of normal

G-rich quadruplex-forming oligonucleotide, alters the ac- cell growth, differentiation and development. Several stud-

tivity of PRMT5 in prostate cancer cells, as demonstrated ies have shown that PRMT5 overexpression is associated

by transcriptional derepression of its target tumor sup- with hyperproliferation of cancer cells, and that inhibition

pressor genes. Furthermore, the interaction between of its activity leads to derepression of anti-cancer genes and

PRMT5 and nucleolin was reduced in the nucleus and slow growth. The function of PRMT5-induced methylation

increased in the cytosol in cells treated with AS1411, which is manifest through a variety of cellular proteins including

indicates that AS1411 alters the localization of nucleolin- cell surface receptors, signaling molecules, structural com-

associated PRMT5 [63]. Thus, it will be important to test ponents of various organelles and chromatin binding pro-

the efﬁcacy of AS1411 in other cancer cell types known to teins. Of particular interest is the association of PRMT5

overexpress PRMT5, and to determine if it has similar with ATP-dependent chromatin remodeling enzymes. The

growth inhibitory effects that can be exploited clinically. outcome of these interactions seems to be different, in that

PRMT5-induced arginine methylation has been impli- association of PRMT5 with SWI/SNF does not result in

cated in other cellular processes, including assembly of the modiﬁcation of its subunits or biochemical activity; howev-

Golgi apparatus, ribosome biogenesis and RNA-mediated er, interaction of PRMT5 with NuRD leads to MBD2

gene silencing (Figure 2a,b). In an attempt to provide a methylation and decreases its afﬁnity for methylated

better understanding of the cytoplasmic function of DNA. By interacting with two distinct chromatin remodel-

PRMT5, interacting partners were identiﬁed by biochemi- ing complexes, PRMT5 can have a broader effect on target

cal puriﬁcation [8]. In addition to MEP50, GM130, which is gene regulation, and in the case of SWI/SNF, PRMT5

a Golgi matrix protein involved in reassembly of the Golgi epigenetically modiﬁes promoter histones at target tumor

complex after cell division, was identiﬁed as a PRMT5- suppressor and cell-cycle regulatory genes, and hence

associated protein and shown to co-localize with PRMT5, causes their transcriptional silencing. Association of

as well as other Golgi markers. PRMT5 methylated GM130 PRMT5 with NuRD has also been shown to have a silenc-

at R6, R18 and R23, and substitution of these sites to ing effect on a CDKN2A; however, biochemical characteri-

lysines affected Golgi apparatus ribbon formation. When zation of methylated NuRD indicates that the complex

PRMT5 was knocked down, the fraction of fragmented does not bind methylated DNA very efﬁciently, which

Golgi apparatus increased, which suggests that PRMT5 raises the possibility that in vivo certain silenced target

impacts fusion of Golgi membranes by methylating GM130 genes might become transcriptionally derepressed. Direct

and inﬂuencing its association with other structural pro- involvement of PRMT5 in epigenetic silencing of E-cad-

teins of the Golgi apparatus [8]. It has also been shown that herin provides additional evidence that altered expression

PRMT5 interacts with and methylates ribosomal protein of PRMT5 impacts cancer cell proliferation and metastasis.

S10 (RPS10), a component of the ribosome 40S subunit [7]. Therefore, these studies clearly demonstrate that the as-

Methylation of RPS10 seems to be crucial for ribosome sociation of PRMT5 with ATP-dependent remodeling com-

assembly, protein synthesis and cell proliferation. Muta- plexes promotes cell survival.

tions that affect methylated arginine residues result in an PRMT5 plays additional essential roles in growth con-

unstable protein that is inefﬁciently incorporated into trol via participation in growth and death-inducing signals

ribosomes. Moreover, RPS10 knockdown cells grow more involving EGFR signaling and TRAIL-induced cell death.

slowly than control cells, and re-expression of wild-type, Based on these ﬁndings, altering of PRMT5 levels or

but not mutant, RPS10 restores cell proliferation, which catalytic activity might facilitate therapeutic manipula-

thus emphasizes the importance of PRMT5-induced meth- tion of these important cellular circuits. Although high-

ylation in ribosome biogenesis and growth control. throughput screens have identiﬁed PRMT inhibitors, as

PRMT5 methylates another group of small RNA-bind- yet there is no evidence that these compounds speciﬁcally

ing proteins called Piwi that are exclusively present in target individual PRMTs [16,65]. Therefore, there is a

germ cells and interact with a speciﬁc class of small non- great need for better and more speciﬁc PRMT inhibitors.

coding RNAs called Piwi-interacting RNAs (piRNAs). In Another area that remains underexplored is the charac-

mouse embryonic germ cells, members of the Piwi family terization of enzymes that remove arginine methylation

repress transcription of transposable elements by inducing marks. Since the discovery of peptidyl arginine deiminase

de novo methylation of their DNA sequences. Biochemical type IV (PADI4), an enzyme capable of converting methy-

puriﬁcation of Piwi-interacting proteins led to the identiﬁ- larginine to citrulline [66,67], there has been little progress

cation of several Tudor-domain-containing proteins, as in understanding how methylarginine marks are erased.

well as PRMT5 and MEP50. Some of the Piwi proteins Several proteins that contain the Jumonji C domain, which

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Review Trends in Biochemical Sciences xxx xxxx, Vol. xxx, No. x

21 Yang, M. et al. (2009) Caenorhabditis elegans protein arginine

is predicted to erase mono- and di-methyl arginine marks

methyltransferase PRMT-5 negatively regulates DNA damage-

without altering the protein sequence, exist. However, of

induced apoptosis. PLoS Genet. 5, e1000514

these proteins, only JMJD6 has been shown to possess

22 Hsu, J.M. et al. (2011) Crosstalk between Arg 1175 methylation and

demethylation activity [68], and this ﬁnding has recently Tyr 1173 phosphorylation negatively modulates EGFR-mediated ERK

been questioned [69,70]. A better understanding of how activation. Nat. Cell Biol. 13, 174–181

23 Liu, F. et al. (2011) JAK2V617F-mediated phosphorylation of PRMT5

PRMT5-induced arginine methylation controls cell prolif-

downregulates its methyltransferase activity and promotes

eration will therefore require characterization of its an-

myeloproliferation. Cancer Cell 19, 283–294

tagonistic arginine demethylases. Furthermore, additional

24 Tanaka, H. et al. (2009) PRMT5, a novel TRAIL receptor-binding

high-throughput screens to identify speciﬁc modulators of protein, inhibits TRAIL-induced apoptosis via nuclear factor-kappaB

both PRMTs and arginine demethylases will probably be activation. Mol. Cancer Res. 7, 557–569

25 Pal, S. et al. (2004) Human SWI/SNF-associated PRMT5 methylates

invaluable in the race to ﬁnd more effective drugs that can

histone H3 arginine 8 and negatively regulates expression of

stop cancer cell growth.

ST7 and NM23 tumor suppressor genes. Mol. Cell. Biol. 24,

9630–9645

Acknowledgment 26 Wang, L. et al. (2008) Protein arginine methyltransferase 5 suppresses

Work in our laboratories was supported by National Institutes of Health the transcription of the RB family of tumor suppressors in leukemia

grants DK084287 to A.N.I. and S.S., CA116093 to S.S., and GM56244 to and lymphoma cells. Mol. Cell. Biol. 28, 6262–6277

A.N.I. 27 Pal, S. et al. (2007) Low levels of miR-92b/96 induce PRMT5 translation

and H3R8/H4R3 methylation in mantle cell lymphoma. EMBO J. 26,

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