identification of methylated proteins by protein arginine n-methyltransferase 1, prmt1, with a new...

Identification of methylated proteins by protein arginine

N-methyltransferase 1, PRMT1, with a new expression cloning strategy

Kazuhiro Wada, Koichi Inoue, Masatoshi Hagiwara*

Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8510, Japan

Received 13 December 2001; received in revised form 11 March 2002; accepted 4 April 2002

Abstract

Methylation at arginines has recently come to attention as a posttranslational modification of proteins, which is implicated in processes

from signaling, transcriptional activation, to mRNA processing. Here we report that several proteins extracted from HeLa cells were

methylated by PRMT1 (protein arginine N-methyltransferease 1) even on a nitrocellulose membrane, while proteins from Escherichia coli are

not methylated with this protein. Screening PRMT1 substrates from a Egt11-HeLa cDNA library, we found that more than half of the 48

cDNA clones obtained encode putative RNA-binding proteins that have RGG (arginine–glycine–glycine) motifs, such as hnRNP R

(heterogeneous nuclear ribonucleoprotein R) and hnRNP K. We cloned two novel arginine methylation substrates, ZF5 (zinc finger 5) and

p137GPI (GPI-anchor protein p137), which do not possess typical RGG motifs. We also cloned a novel protein that has RGG motifs, but

does not have any other RNA-binding motifs. We tentatively termed this clone SAMT1 (substrate of arginine methyl transferase 1).

A63-VLD-65 to AAA mutation of PRMT1 suppressed the methylation of recombinant SAMT1 and other RGG proteins in the HeLa extracts.

This systematic screening of substrate proteins with the solid phase methylation reaction will contribute to identify new roles of PRMT

family. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Arginine methylation; PRMT1; RGG motif; RNA-binding protein; Solid phase screening

1. Introduction

Methylation of arginine residues by covalent modifica-

tions occurs in eukaryotic proteins during or shortly follow-

ing mRNA to protein translation. Evidence for posttransla-

tional methylation of arginine residues was first provided by

the presence of radioactive species chromatographing at

positions near that of arginine in acid hydrolysates of

isolated calf thymus nuclei proteins incubated with [meth-

yl14C]-S-adenosyl-L-methionine (SAM) [1]. The products of

the methylation reaction were determined to be NG-mono-

methylarginine and NG,NG-dimethylarginine [2]. Protein

arginine N-methyltransferases (PRMTs) catalyze the

sequential transfer of methyl groups from SAM to the

guanidino nitrogens of arginine residues within proteins

[3,4]. It now appears that there are at least two distinct

classes of protein arginine methyltransferases according to

their substrate specificity and reaction products [4]. The

type I enzymes catalyze the formation of NG-monomethy-

larginine and asymmetric NG,NG-dimethylarginine residues,

while type II enzymes catalyze the formation of NG-mono-

methylarginine and symmetric NG,NVG-dimethylarginine.

The asymmetric dimethylarginine appears to be the more

common form while symmetric dimethylarginine has so far

been identified only in a few proteins, including myelin

basic protein (MBP) [5], and spliceosomal Sm proteins D1

and D3 (SmD1 and SmD3) [6]. Reported type I enzymes are

the yeast Hmt1p/Rmt1p [7] and the mammalian PRMT1 [8],

PRMT3 [9], and CARM1/PRMT4 [10]. PRMT1 and yeast

Hmt1p/Rmt1p prefer to methylate arginine residues in

glycine rich regions, which are found in several RNPs,

fibrillarin, and nucleolin [11]. However, CARM1/PRMT4

has little or no activity with substrates preferred by these

enzymes, and the best substrate reported for CARM1/

PRMT4 is histone H3 [10]. JBP1/PRMT5 can methylate

histone H2A and H4 and myelin basic protein (MBP) [11].

Recently, a potential candidate for a type II enzyme, JBP1/

PRMT5, has been discovered in mammalian cells with an

apparent homologue in yeast, Hsl7 [12]. Yet, its sequence is

well conserved with other family members, the catalytic

activity of PRMT2 has not yet been demonstrated [13]. The

conservation among protein arginine N-methyltransferases

0167-4889/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0167 -4889 (02 )00202 -1

* Corresponding author. Tel.: +81-3-5803-5836; fax: +81-3-5803-5853.

E-mail address: [email protected] (M. Hagiwara).

www.bba-direct.com

Biochimica et Biophysica Acta 1591 (2002) 1–10

may underscore the potential biological importance of this

posttranslational modification. In addition to these, the type

III enzyme was also recently discovered in yeast, and this

catalyzes the monomethylation of the internal y-guanidinonitrogen atom of arginine residues [14].

PRMT1 knockout mouse has already been established,

and the phenotype of the homozygous null mutant is

embryonic lethal [15], suggesting that PRMT1 is required

for very early stages of mouse development. Though recent

evidence has implicated arginine-specific protein methyl-

transferases in nuclear export of some hnRNP proteins and

in several signaling pathways (reviewed in Ref. [4]), spe-

cific roles of protein methylation and the relevant protein

substrates have not been identified. Because each methyl-

transferase recognizes a different set of protein substrates,

systematic analysis of their substrate specificity is now

required for differentiating their function. To this end, we

report here a new expression cloning strategy to identify

substrate proteins of PRMT1, which is potentially applica-

ble to isolate substrates specific for each member of the

PRMT family.

2. Materials and methods

2.1. In vitro methylation reaction

In vitro methylation assay was performed as described by

Najbauer et al. [16] with a few modifications. One micro-

gram each of recombinant proteins was added in a 20 Alreaction mix containing 50 mM Na-morpholinepropanesul-

fonic acid [MOPS] (pH 7.2), 300 mM, NaCl, 2 mM ethyl-

enediaminetetraacetic acid [EDTA], 1 mM phenylmethyl-

sulfonyl fluoride [PMSF], 0.1 Ag/ml leupeptin and 0.4 AM S-

adenosyl-L-[methyl-3H] methionine [3H-SAM] (specific

activity of 1.3 Ci/mmol; Amersham Biosciences, NJ) with

0.02–0.04 Ag/Al recombinant PRMT1 or bovine serum

albumin [BSA] (as a negative control) and incubated at 30

jC for 45 min. The methylation reaction was terminated by

adding 20 Al sodium dodecyl sulfate [SDS] sample buffer and

boiled for 5 min. Each 20 Al reaction solution was then loadedonto a 15% SDS-polyacrylamide gel, and separated by

electrophoresis. Gels were stained with Coomassie blue R-

250 to visualize protein bands and then fluorographed, dried

and exposed to Kodak film at � 80 jC for 2 days.

2.2. Solid phase methylation assay

Cell extracts were size-separated on SDS-polyacrylamide

gel electrophoresis [SDS-PAGE] and transferred to a nitro-

cellulose membrane. The membrane was then incubated in a

reaction mix containing 50 mM Na-MOPS (pH 7.2), 300

mM NaCl, 2 mM EDTA, 1 mM PMSF, 0.1 Ag/ml leupeptin,

and 0.4 AM 3H-SAM with 1 Ag of recombinant PRMT1 at

30 jC for 1 h, washed with PBS [phosphate-buffered saline]

and TPBS [Tris–phosphate-buffered saline], fluorographed,

dried, and exposed to Kodak film at � 80 jC for 2 days.

2.3. Screening of a cDNA library by solid-phase methylation

The HeLa UNI-ZAP cDNA expression library (Strata-

gene, CA) was plated on Escherichia coli XL-1 blue strain

Fig. 1. Detection of methylated proteins by PRMT1 on a nitrocellulose membrane. (A) Nuclei extracts and cytosolic fractions prepared from HeLa cells

cultivated for 3 days in either the absence (lanes 1 and 2) or presence (lanes 3, 4, 5, and 6) of the methyltransferase inhibitor adenosine dialdehyde (AdOX),

blotted on a nitrocellulose membrane, and incubated with (lanes 1, 2, 3, and 4) or without (lanes 5 and 6) PRMT1. (B) Whole E. coli proteins from IPTG-

induced GST-hnRNPA1 were separated on SDS-PAGE and transferred on a nitrocellulose membrane (lanes 2, 4 and 6). IPTG-untreated E. coli proteins were

similarly prepared as negative controls (lanes 1, 3, and 5). The nitrocellulose membranes were incubated in the absence (lane 1 and 2) or presence (lanes 3 and

4) of recombinant PRMT1 in methylation reaction buffer including 3H-SAM at 30 jC for 1 h. The protein amount of each lane was normalized as shown in the

Coomassie staining (lanes 5 and 6).

K. Wada et al. / Biochimica et Biophysica Acta 1591 (2002) 1–102

at a density of 1.8� 104 plaques per 100� 140 mm square

agar plate. After incubation for 3 h at 42 jC, the plates wereoverlaid with nitrocellulose membrane filters (PROTRAN,

Schleicher & Schuell, NH) that had been soaked with

10 mM isopropyl-h-D-thiogalactopyranoside [IPTG]. The

plates were incubated at 37 jC for another 4.5–5 h, and

their filters were peeled off, immersed in blocking solution

[5% skim milk in PBS] and gently agitated at 4 jC for 1 h.

Fig. 2. Expression cloning protocol of substrate of PRMT1. (A) Brief protocol of the screening of PRMT1 substrates from a kgt 11-HeLa cDNA library. (B)

Representative result of the first screening (upper panel). Positive clones indicated by arrows (I, II, and III of the upper panel) were further condensed to

monoclonal plaques through a second (not shown) and third screening (lower panels).

Table 1

Summary of expression screening for PRMT1 substrates

K. Wada et al. / Biochimica et Biophysica Acta 1591 (2002) 1–10 3

The filters were then washed three times for 20 min in PBS,

incubated for 30 min at 4 jC in the methylation buffer (50

mM Na-MOPS [pH 7.2], 2 mM EDTA, 0.3 M NaCl, 0.1 Ag/ml leupeptin, 1 mM PMSF, 0.4 AM 3H-SAM; specific

activity of 1.3 Ci/mmol) with 0.02–0.04 Ag/Al of recombi-

nant PRMT1 and further incubated at 30 jC for 2–3 h for

the solid-phase methylation with gentle agitation. The filters

were then washed with TPBS three times for 20 min and air

dried. The filters were fluorographed with EN3HANCE

spray (Dupont, DE), dried and exposed to X ray film

Fig. 3. Primary structure and expression of SAMT1. (A) Predicted amino acid sequence of SAMT1. RGG motifs are indicated by black lines. (B) Expression of

SAMT1 in various human tissues. Human multiple tissue Northern blot I or II (Clontech) was hybridized with a probe of full-length SAMT1 cDNA. The

tissues from which the RNA is derived are indicated above and size standards (in kb) are indicated to the left.


(BioMax MR-2, Kodak, NY) at � 80 jC for 1 week. At the

first screening, 607 positive clones were detected in

2.0� 105 plaques. We picked up 100 of these clones and

succeeded to isolate 83 positive clones by the third screen-

ing. Positive clones were converted to plasmids with the

Exassist/Solr E. coli system (Stratagene).

2.4. Northern hybridization analysis

SAMT full-length cDNA probe or h-actin probe was

labeled with radioactive [a-32P] dCTP using the multi-

prime DNA labeling system (Amersham Biosciences). A

human multiple tissue Northern blot (Clontech, CA)

membranes were used. Hybridization was carried out at

42 jC overnight in hybridization mixture (6� SSC [1�SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 50%

formamide, 1% SDS, 1� Denhardt’s solution, 10%

dextran sulfate, 100 Ag of denatured salmon sperm DNA

per ml). The membrane was washed three times at room

temperature in washing buffer (0.1� SSC, 0.1% SDS),

and the hybridized transcripts were observed with a

BAS2000 image analyzer (Fuji Film, Tokyo).

2.5. Cell culture and transfection assays

HeLa cells were grown on cover slips in Dulbecco’s

modified Eagle’s medium [DMEM] supplemented with

10% calf serum, 100 U/ml penicillin, and 100 U/ml

streptomycin. Expression vectors of pGFP (Clontech)

containing cloned full-length cDNAs were introduced into

cells at approximately 60% confluence with Lipofect-

AMINE (Life Technologies, NY) following the trans-

fection conditions as recommended by the manufacturer.

For immunoflourescence, cells were washed in PBS and

fixed for 15 min with 4% paraformaldehyde in PBS at

room temperature, permeabilized, and blocked in PBS con-

taining 0.4% Triton X-100, 1.5% BSA, and 5% normal goat

serum.

3. Results

3.1. Detection of methylated proteins by PRMT1 on a

nitrocellulose membrane

First we examined whether proteins in HeLa cell

extracts could be methylated by PRMT1 on a nitro-

cellulose membrane. HeLa cells were cultivated for 3

days in the presence of the methyltransferase inhibitor, 20

AM adenosine dialdehyde (AdOX), to prevent endoge-

nous methylation that would mask our assay. Nuclei and

cytosolic fractions were then size-separated on SDS-

PAGE, transferred to a nitrocellulose membrane, and

incubated with recombinant human PRMT1 and the

methylation substrate 3H-SAM at 30 jC for 30 min.

Four major bands and seven minor bands in the nuclei

fraction appeared after 2 days exposure (Fig. 1A, lane 3),

whereas four major bands and three minor bands

appeared in the cytosolic fraction (Fig. 1A, lane 4). All

these bands were faint or absent in extracts prepared

from HeLa cells cultivated without AdOX (Fig. 1A, lanes

1 and 2), suggesting that most arginines in these proteins

were already methylated in vivo in HeLa cells. To further

examine the validity of the in vitro methylation reaction

on the nitrocellulose membrane, we methylated recombi-

nant hnRNP A1 under the control of an IPTG promoter

in E. coli. Proteins were isolated from IPTG-treated or

-untreated whole E. coli containing recombinant hnRNAP

A1, separated on SDS-PAGE and transferred to a nitro-

cellulose membrane. Methylated proteins were specifically

Fig. 3 (continued).


detected in the IPTG-induced E. coli containing hnRNP

A1 (Fig. 1B, lanes 3 and 4). In E. coli not containing

human hnRNP A1, no positive band was found in IPTG-

treated E. coli (Fig. 1B, lanes 1 and 2). These results

show in vitro methylation by PRMT1 is specific to the

human protein and that E. coli does not methylate the

human protein, at least at the proprietorial arginine

residues. These results prompted us to perform a com-

prehensive substrate screening of PRMT1 using a Egt11-HeLa cDNA expression library in E. coli host with

IPTG-induced cDNA protein products separated on the

nitrocellulose membrane.

3.2. Expression screening of PRMT1 substrates

Fig. 2 shows the experimental order of the screening

(Fig. 2A) and a representative of the first and third

screening (Fig. 2B). After the first screening, 607 positive

clones were detected in 2.0� 105 plaques. We picked up

100 of these clones and succeeded to isolate 83 positive

clones by the third screening. We determined the nucleo-

tide sequences of 48 of these clones. A BLAST search

[17] against GeneBank revealed that more than half of

the clones encode putative RNA-binding proteins such as

hnRNP R, hnRNP K, GRY-RBP (glycine, arginine, and

tyrosine-rich RNA binding protein), TAFII68 (TBP asso-

ciated factor II68), RBP56 (RNA binding protein 56), and

TLS/FUS (translocated in liposarcoma), as summarized in

Table 1. A common feature of these proteins is to contain

RGG motifs at their C-terminus (indicated by stripes in

the right end column of Table 1). Eight clones encoded

the same novel protein, which also has RGG motifs at its

C-terminus, but not other RNA-binding motifs. We

termed this clone SAMT1 (substrate of arginine methyl-

transferase 1). The predicted amino acid sequence of

SAMT1 is shown in Fig. 3A. During preparation of this

manuscript, a clone named CG1-55 coding the identical

protein to SAMT1 was registered to the database of

GeneBank, as one of conserved human genes by com-

parative proteomics [18]. Northern blot analysis of

SAMT1 mRNA showed ubiquitous expression in human

tissue samples (Fig. 3B). We tagged cloned cDNAs with

green fluorescent protein (GFP) and GFP-tagged SAMT1

was predominantly localized to the cytoplasm around

nucleus, presumably at the endoplasmic reticulum (ER),

while the other RGG-proteins were localized in the

nucleus in HeLa cells (Fig. 4). Other five clones encode

a transcription factor, ZF5 (zinc finger 5) and a GPI

(glycosyl phosphatidylinositol)-anchor protein, p137GPI.

Both of these proteins do not have typical RGG motifs.

3.3. Methylation of cloned proteins by PRMT1 in vitro

To determine whether the cloned proteins are really

methylated by PRMT1 in vitro, we prepared recombinant

proteins of GST (glutathione sulfatransferase)-ZF5, GST-

p137GPI, GST-hnRNP R, GST-GRY-RBP, GST-TAFII68,

GST-TLS, and GST-SAMT1 (Fig. 5A). These proteins

Fig. 4. Intracellular localization of cloned proteins. HeLa cells were transfected with the expression vectors, fixed, and analyzed by indirect immunofluorescence

staining. PRMT1was expressed as a fusion protein with the HA epitope and stained with an anti-HAmonoclonal antibody (a). The cloned RGG proteins, hnRNP

R (b), GRY-RBP (c), TLS (d), TAFII68 (e), and SAMT1 (f) were tagged with GFP protein and stained with anti-GFP monoclonal antibody. Fluorescein

isothiocyanate-labeled anti-mouse second antibody was used to visualize each protein. Scale bar in (a): 20 Am.


were then incubated at 37 jC for 30 min with recombi-

nant PRMT1 and 3H-SAM. All of above proteins were

highly methylated by PRMT1, while GST alone was not

methylated (Fig. 5A). These proteins were methylated

when they were incubated with HeLa cell extracts and 3H-

SAM (Fig. 5C). Next, we tested the effect of a dominant-

negative PRMT1 on methylation of HeLa cell extracts. We

mutated PRMT1 cDNA by substituting alanines for three

amino acids, valine 63, leucine 64, and aspartic acid 65,

located in the highly conserved region that is proposed to be

important for SAM binding and thus for methytlransferase

activity [10] (Fig. 5B). The VLD to AAA mutation of

PRMT1 abolished the enzymatic ability to methylate hnRNP

A1 in vitro (data not shown). Administration of the mutant

PRMT1 suppressed the methylation of GST-hnRNP A1,

GST-TAFII68, and GST-SAMT1 by the extract (Fig. 5C),

suggesting that these proteins are methylated mainly by

PRMT1 in the HeLa cell extract.

4. Discussion

Protein function is often modulated by posttranslational

modifications; phosphorylation, acetylation, glycosylation,

and methylation. Though an important role for protein

methylation in bacterial chemotaxis has long been appreci-

ated (reviewed in Ref. [19]), a role of protein methylation in

signal transduction has not been explored in mammalian

cells with the same breadth and intensity that protein

phosphorylation has enjoyed in recent years. Unlike phos-

Fig. 5. Methylation of cloned proteins by PRMT1 in vitro. (A) Recombinant proteins of GST-hnRNP R (lane 1), GST-GRY-RBP (lane 2), GST-TAFII68 (lane 3),

GST-TLS (lane 4), GST-SAMT (lane 5), GST-ZF5 (lane 6), and GST-GPI anchor protein (lane 7) were purified from E. coli and incubated with [methyl3H]-SAM

in the presence of recombinant PRMT1. GSTwas used as a negative control (lane 8). Methylation of each protein was detected by autoradiography (upper panels)

and protein amount of each methylation reaction was confirmed with Coomassie staining (arrow heads of lower panels). (B) The 63-VLD-65 to AAA mutation of

PRMT1. (C) GST-hnRNPA1, GST-TAFII68 and GST-SAMT1 were incubated with HeLa cell extract, 3H-SAM, and indicated amount of PRMT1 VLD to AAA

mutant at 30 jC for 1 h. The amount of the RGG proteins were normalized by Coomassie staining as shown in the lower panels.


phorylation, protein methylation might be irreversible,

because protein demethylases have not been discovered so

far [20]. Yet, this may be a key mechanism of cell function

as a growing number of proteins are being found to contain

methylated amino acids, monomethylarginine and asymmet-

ric dimethylarginine. Many of these proteins are associated

with RNA, such as the heterogeneous nuclear ribonucleo-

protein (hnRNP) A1 [21,22], the U3 small nuclear RNA-

binding protein fibrillarin [23], the rRNA binding protein

nucleolin [24], poly(A)-binding protein (PAB) II [25], the

Src-associated substrate, Sam68 [26], interleukin enhancer

binding factor3 [27], and the yeast Npl3 protein [28,29]. As

a result, the hnRNPs contain about 65% of the total NG,NG-

dimethylarginine found in the HeLa cell nucleus [11].

The site of arginine methylation for type I enzymes

typically occurs within glycine- and arginine-rich (GAR)

domains from which similar consensus sequences have been

derived [4,7,16,30]. A search of the GenPept database for a

consensus FGGRGGF motif revealed 13 substrate candi-

dates [16]. Of these, 10 were known or presumed to interact

with RNA [16]. In our expression screening, we did not

isolate any of 13 candidates, and seven of cloned 9 genes

encode hnRNP R, hnRNP K, GRY-RBP, TAFII68, RBP58,

TLS/FUS, and SAMT1, respectively, all of which contain

the consensus sequence or consensus-like sequences with

two to three amino acid substitutions (Table 2). However,

the other two do not have the consensus motif. Recently,

Smith et al. [25] reported that most of the methylated

arginine residues were found in Arg-X amino acid-Arg

(RXR) clusters in the C-terminus of PAB2. They showed

both PRMT1 and PRMT3 methylated the arginine in the C-

terminal domain. As shown in Table 2, ZF5 and p137GPI

contained the RXR sequence, though the methylation sites

of these proteins have not been identified yet.

The glycine-rich clusters with many glycine–glycine

dipeptides interspersed with aromatic or arginine residues

are termed ‘‘RGG’’ [31]. The RGG motif is ubiquitous in

proteins involved in diverse aspects of RNA metabolism

such as pre-rRNA processing factors, pre-mRNA splicing

factors, hnRNPs, and RNA helicases [32]. The C-terminal

RGG motif of hnRNP U, which lacks RRM (RNA recog-

nition motif), facilitate as the RNA-binding motif [31]. In

nucleolin, which has two RRMs, the C-terminal RGG

domain mediates RNA binding even without the RRMs

[31]. However, Bouvet et al. [33] reported that this domain

interacts with 10 ribosomal proteins. Likewise, in hnRNP

A1, RGG domain is not only involved in interaction with

RNA [34] but also with proteins [35]. In spite of prevalence

of RNA binding proteins with RGG motifs, few studies

have directly addressed the effect of arginine methylation on

binding of target proteins to RNA. However, recent studies

have examined arginine methylation in modulating protein–

Table 2

(F/G)GGRGG(F/G) homologous sequences and RXR sequences found in cloned proteins

Gene (F/G)GGRGG(F/G) homologous

sequence

RXR sequence Gene (F/G)GGRGG(F/G) homologous

sequence

RXR sequence

hnRNP R 447aa-GRGRGGGRGGY-458aa 106aa-RQR-108aa RBP58 203aa-GGDRGGF-209aa 185aa-RGR-187aa

500aa-RGRGGGRGGRG-509aa 306aa-RRR-308aa 334aa-RRGRGGYRGRGGFQ- 395aa-RGR-397aa

540aa-RGSRGGRGGP-549aa 516aa-RGR-518aa GRGGD-352aa

562aa-RGNRGGN-568aa 524aa-RGR-526aa 405aa-YRGRGGRGGDRGGY-418aa

554aa-RGR-556aa 456aa-GGDRGGG-462aa

GRY-RBP 445aa-GRGRGGRGGY-455aa 103aa-RQR-105aa 464aa-GGDRGGG-470aa

495aa-ARGRGGR-501aa 303aa-RRR-305aa 472aa-GGDRGGG-478aa

536aa-RGARGGA-541aa 510aa-RGR-512aa 480aa-GGDRGGY-486aa

551aa-RGARGGRGGN-560aa 518aa-RGR-520aa 487aa-GGDRGGG-493aa

556aa-RGR-558aa 495aa-GGDRGGY-501aa

hnRNP K 255aa-MRGRGGF-261aa 35aa-RSR-37aa 502aa-GGDRGGY-508aa

296aa-RGGRGGS-302aa 326aa-RGR-328aa 509aa-GGDRGGY-515aa

TAFII 68 200aa-GGDRGGF-206aa 182aa-RGR-184aa 516aa-GGDRGGY-522aa

331aa-RRGRGGYRGRGGF-343aa 392aa-RGR-394aa 532aa-GGDRGGG-538aa

400aa-GYRGRGGRGGDRG- 523aa-RSR-525aa 559aa-GGDRGGG-565aa

GYGG-417aa 567aa-GGDRGGY-573aa

453aa-GGDRGGG-459aa TLS/FUS 241aa-PRGRGGGRGGRGGM-254aa 216aa-RGR-218aa

461aa-GGDRGGG-467aa 380aa-GNGRGGRGRGGP-319aa

469aa-GGDRGGG-475aa 404aa-GGGRGGF-410aa

467aa-GGDRGGY-483aa 473aa-RGGRGGY-479aa

484aa-GGDRGGG-490aa 484aa-YRGRGGDRGGFRGGRGG-

492aa-GGDRGGY-498aa GDRGGF-506aa

499aa-GGDRGGY-505aa SAMT1/CGI-55 162aa-IRGRGGL-168aa 134aa-RER-136aa

506aa-GGDRGGY-512aa 169aa-GRGRGGR-175aa

513aa-GGDRGGY-519aa 364aa-RPGRGGRGGRGGRGR-

528aa-GGDRGGG-535aa GGR-382aa

556aa-GGDRGGG-562aa Zinc finger 5 (� ) 291aa-RLR-293aa

564aa-GGDRGGY-570aa 382aa-RHR-384aa

p137GPI (� ) 303aa-RQR-305aa


protein interactions (reviewed in Ref. [36]). Asymmetric

arginine methylation of Sam68 within the RGG domains

decreases Sam68 binding to SH3 proteins but not to WW

domain-containing proteins, suggesting that methylation

may alter the specific protein–protein contacts [37]. Sim-

ilarly, methylation of STAT1 (signal transducer and activator

of transcription) decreases its affinity for its inhibitor PIAS1

[38]. Recently, we screened the factors that interact with the

RGG domain of Npl3p with the two hybrid system and

isolated nine different genes of which five have RGG

domains [39].

Disruption of Hmt1p/Rmt1p in yeast showed that the

methyltransferase activity is critical for efficient nuclear

transport of the specific hnRNP proteins, Npl3p, and Hrp1p,

and is important in RNA metabolism [40]. Npl3p is also

modulated by phosphorylation in the arginine/serine (RS)

domain by Sky1p and the phosphorylation influences the

shuttling of Npl3p [41,42], indicating the complex regula-

tion of hnRNP proteins with double modulation of arginine

methylation and serine phosphorylation. In mammals,

mouse embryonic stem cells bearing a null mutation of

PRMT1 were viable, though the mouse was embryonic

lethal [15]. However, the yeast Hmt1p/Rmt1p deletion strain

was viable under standard growth condition [40]. In support

of this, transfected HeLa cells with the VLD to AAA

mutant, the dominant-negative PRMT1, expression vector

survived and showed normal growth (unpublished data).

Desrosiers and Tanguay [43] and Najbauer et al. [44]

reported that protein methylation is involved in cellular

stress responses and the aging/repair of proteins. Consider-

ing that PRMT1 is the predominant type I arginine methyl-

ase in mammalian cells [9], PRMT1 may be required for

specific developmental processes or for stress responses.

Systematic screening of substrate proteins with the solid

phase methylation reaction described here will contribute to

identify new roles of PRMT family.

Acknowledgements

Part of this work was performed in SEEDS laboratory of

Yamanouchi Pharmaceutical Co., Ltd. for the Industrial

Science and Technology Program supported by New Energy

and Industrial Technology Development (NEDO). This work

was partially supported by Research for the Future Program,

the Inamori Foundation, Nissan Science Foundation, The

Mitsubishi Foundation, and Grants from the Ministry of

Education, Science and Culture in Japan. We thank H.

Hattou, T. Urushido, and T. Ishiguro for technical supports

and Dr. Erich D. Jarvis for editing and critical comments.

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identification of methylated proteins by protein arginine n-methyltransferase 1, prmt1, with a new...

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