From the Cell Biology Laboratory, Korea Research
Institute of Bioscience and Biotechnology, Yusong, Taejeon 305-333 and
the ¶ Biomolecule Research Team, Korea Basic Science Institute,
Taejeon 305-333, Republic of Korea
Received for publication, September 21, 2000, and in revised form, December 21, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We found that JBP1, known as a human
homolog (Skb1Hs) of Skb1 of fission yeast, interacts with NS3 of the
hepatitis C virus in a yeast two-hybrid screen. Amino acid sequence
analysis revealed that Skb1Hs/JBP1 contains conserved motifs of
S-adenosyl-L-methionine-dependent protein-arginine methyltransferases (PRMTs). Here, we demonstrate that
Skb1Hs/JBP1, named PRMT5, is a distinct member of the PRMT family.
Recombinant PRMT5 protein purified from human cells methylated myelin
basic protein, histone, and the amino terminus of fibrillarin fused to
glutathione S-transferase. Myelin basic protein
methylated by PRMT5 contained monomethylated and dimethylated arginine
residues. Recombinant glutathione S-transferase-PRMT5
protein expressed in Escherichia coli also contained the
catalytic activity. Sedimentation analysis of purified PRMT5 on a
sucrose density gradient indicated that PRMT5 formed distinct
homo-oligomeric complexes, including a dimer and tetramer, that
comigrated with the enzyme activity. The PRMT5 homo-oligomers were
dissociated into a monomer in the presence of a reducing agent, whereas
a monomer, dimer, and multimer were detected in the absence or at low
concentrations of a reducing agent. The results indicate that both
covalent linkage by a disulfide bond and noncovalent association are
involved in the formation of PRMT5 homo-oligomers. Western blot
analysis of sedimentation fractions suggests that endogenous PRMT5 is
present as a homo-oligomer in a 293T cell extract. PRMT5 appears to
have lower specific enzyme activity than PRMT1. Although PRMT1 is known
to be mainly located in the nucleus, human PRMT5 is predominantly
localized in the cytoplasm.
Protein arginine methylation is an irreversible,
post-translational covalent modification. Protein-arginine
methyltransferases (PRMTs)1
transfer the methyl group from
S-adenosyl-L-methionine to the guanidino
nitrogen atoms of an arginine residue (1). PRMTs are classified into
two major types, I and II, based on substrate and reaction product
specificity. Both type I and II PRMTs are common in the formation of
monomethylarginine, but the two differ in that type I PRMT catalyzes
asymmetric dimethylarginine, whereas type II PRMT produces symmetric
dimethylarginine. Type I PRMTs methylate arginines in the
Arg-Gly-Gly-rich region, known as the RGG motif, present in many
RNA-binding proteins (2-4), or in the Arg-Xaa-Arg motif in
poly(A)-binding protein II (5). Myelin basic protein (MBP) and the
spliceosomal D1 and D3 proteins are the only known in vivo
substrates for type II PRMT (6-8). However, the classification of type
I and II protein-arginine methyltransferases is tenuous because they
are based only on substrate and reaction product specificity
(1).
Although five different kinds of genes for protein-arginine
methyltransferases PRMT1, HRMT1L1 (human
arginine
methyltransferase-1 L1)/PRMT2, PRMT3, CARM1 (coactivator-associated
arginine
methyltransferase-1)/PRMT4, and Skb1Hs
(Shk1 kinase-binding
protein-1 Homo
sapiens)/JBP1 (named PRMT5) have been
cloned in mammalian cells, protein-arginine methyltransferase
activities of the gene products are demonstrated in only PRMT1, PRMT3,
and CARM1/PRMT4 (9-11). The gene for rat PRMT1 is the first mammalian
gene cloned 30 years after the discovery of protein arginine
methylation (12, 13). PRMT1 interacting with the mammalian
immediate-early protein (TIS21/PC3), known as BTG2 (14, 15), is a
predominant protein-arginine methyltransferase in mammalian cells and
tissues (16, 17). Subsequently, human PRMT1, which is almost identical
to rat PRMT1, was found to be associated with the intracytoplasmic
domain of the interferon- Independently, we found that NS3 (nonstructural
protein-3) of the hepatitis C virus (HCV) interacts with an
Skb1Hs protein, a human homolog of Skb1 (Shk1
kinase-binding
protein-1) of fission yeast, in a yeast
two-hybrid screen. HCV causes acute and chronic liver diseases such as
liver cirrhosis and hepatocellular carcinoma (26, 27). The NS3 protein
of HCV not only contains serine protease (28-31) and RNA helicase (32,
33) activities, both of which appear to be essential for the virus
replication, but also has been implicated in cellular
transformation (34, 35). For example, NIH3T3 mouse fibroblasts
transfected with the N-terminal domain of NS3 become transformed and
are tumorigenic in nude mice (34). In fission yeast, Skb1 interacts
with the Shk1 kinase, which is a yeast homolog of human
p21Cdc42/Rac1-activated kinase (24). Skb1 has been
suggested to regulate mitosis negatively and can be functionally
replaced with its human homolog (Skb1Hs) in fission yeast (25). In this
context, an interaction between Skb1Hs and NS3 may play a role in liver
diseases caused by HCV. To investigate a function of Skb1Hs interacting with the viral protein, its amino acid sequence was compared with those
of genes registered in the GenBankTM/EBI Data Bank. The
comparison revealed that the C-terminal domain of Skb1Hs contains an
extensive homology to a family of proteins with arginine-specific
protein methyltransferase activity.
In this study, we focused on the biochemical properties of PRMT5 and
found that the arginine residue present in MBP is methylated by PRMT5
and that homo-oligomerization is important for the catalytic activity.
A homomeric complex of PRMT5 was detected in vivo by co-immunoprecipitation analysis. The homomeric complex of PRMT5 could
be separated into a dimer and multimer by sucrose gradient sedimentation. Purified PRMT1 also forms homo-oligomers. PRMT5 forms
distinct homo-oligomeric complexes different from those formed by
PRMT1, but both covalent and noncovalent associations are involved in
the homo-oligomerization of PRMT5 and PRMT1. The homo-oligomeric
complexes of PRMT5 and PRMT1, both of which methylate MBP in
vitro, may account for the controversial polypeptide compositions of protein-arginine methyltransferases previously purified from cells
and tissues (3, 36, 37).
Cell Cultures--
293T, COS-1, and Chang liver monolayer
cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin (Life Technologies, Inc.) in an atmosphere of
5% CO2 and air humidified at 37 °C.
Cloning of Human PRMT5--
A yeast two-hybrid screen was
conducted using a HeLa cell cDNA library and a bait plasmid that
encodes the LexA DNA-binding domain fused to the N-terminal domain
(amino acids 1027-1297) of HCV NS3. The isolation of positive clones
and subsequent analysis were carried out as previously reported (38).
One of the positive clones, named clone 4-7, encodes a partial region
of human PRMT5 cDNA. To obtain full-length 4-7, primers and a HepG2
cell cDNA library were used in polymerase chain reactions. A
partial clone missing ~200 base pairs from the N terminus of clone
4-7 was obtained and sequenced. The missing N-terminal portion was
cloned by reverse transcription-polymerase chain reaction of total
mRNAs from HepG2 cells. The full-length PRMT5 cDNA was inserted
into the HincII site of pBluescript KS (Stratagene) and
sequenced, yielding pBS-4-7F.
Plasmids--
pFLAG-PRMT5, a FLAG epitope-tagged expression
plasmid, was constructed by inserting a 1661-base pair
HindIII-XhoI fragment (amino acids 85-637) of
PRMT5 cDNA into the HindIII-SalI fragment of
a pCMV2-FLAG vector (Sigma) and then inserting the remaining 258-base
pair HindIII fragment (amino acids 1-84) of PRMT5 into the
5'-HindIII site. To construct pFLAG-PRMT5-C (where C is the C-terminal region of PRMT5), a 992-base pair
PstI-XhoI fragment (amino acids 308-637) of
pBS-4-7F was subcloned into pUC19, and then the
HindIII-XhoI fragment was subcloned into
pCMV2-FLAG. To make a glutathione S-transferase (GST) fusion
expression plasmid of PRMT5, a full-length fragment of PRMT5 was
inserted into the ClaI-NotI fragment of pEBG
(pGST-PRMT5). An amino-terminal region (amino acids 1-309) of PRMT5
was subcloned into the ClaI-NotI fragment of pEBG
(pGST-PRMT5-N). A C-terminal region (amino acids 315-637) of PRMT5 was
subcloned into the BamHI-NotI fragment of pEBG
(pGST-PRMT5-C). To construct the green fluorescent protein (GFP) fusion
plasmid pGFP-PRMT5, the BamHI-XhoI fragment of
full-length PRMT5 was taken from plasmid pBS-4-7F and subcloned into
the BglII-SalI fragment of pEGFP-C1
(CLONTECH).
To make a methyltransferase domain I mutant
(pGST-PRMT5-M), primers 5'-AAGGATCCACCATGGCGGCGATGGCGGTCGGG-3',
5'- AAGCGGCGGCCTAGAGGCCAATGGTATATGAGCG-3', 5'-AACTCGCGTCGCAGCACCATCAGTACCTGGAC-3', and
5'-AACTCGAGCCCCTGGTGAACGCTTCCCTG-3' were used in the polymerase
chain reactions to amplify a mutant of PRMT5 cDNA. The amplified
fragment was subcloned into the BamHI-NotI fragment of pGEX4T-1 (Amersham Pharmacia Biotech). The mutated region
was confirmed by sequencing. Full-length PRMT5 and the C-terminal
region of PRMT5 (amino acids 315-637) were each cloned into the
BamHI-NotI fragment of pGEX4T-1 (yielding
pGST-PRMT5 and pGST-PRMT5-C, respectively) for the preparation of GST
fusion proteins expressed in Escherichia coli.
To construct a FLAG-tagged PRMT1 expression plasmid, pCMV2-PRMT1
(FLAG-PRMT1), rat PRMT1 cDNA was amplified from
pGEX(SN)-PRMT1 (9) using primers 5'-CCGGATCCACCATGGCGGCAGCCGAGGCCGCG-3'
and 5'-CCGCGGCCGCTCAGCGCATCCGGTAGTCGG-3'. The amplified PRMT1 DNA was subcloned into the BamHI-NotI fragment of
pBluescript KS, digested with HindIII and NotI,
and inserted into the HindIII-NotI fragment of
pCMV2-FLAG.
Protein-arginine Methyltransferase Assay--
293T cells (5 × 105 cells/ml) were plated 18-24 h before transfection.
Expression plasmids (1-10 µg) were used in the transfections. After
36 h, the cells were lysed with lysis buffer (25 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 10 µg/ml aprotinin,
and 10 µg/ml leupeptin). The lysates were cleared by centrifugation
at 12,000 rpm for 10 min. Anti-FLAG antibody- or glutathione-conjugated
agarose beads were added to the cleared lysates and incubated at
4 °C for 2 h with rocking. The beads were washed twice with
lysis buffer and twice with PRMT assay buffer (25 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride). Proteins bound to the
beads or the purified PRMT proteins were then incubated in a 40-µl
reaction volume containing 0.25 µCi of
S-[methyl-14C]adenosyl-L-methionine
([14C]AdoMet; specific radioactivity of 56 mCi/mmol;
Amersham Pharmacia Biotech) and 0.1-5 µg of methyl acceptors at
30 °C for 2 h. Methylation reactions were stopped by the
addition of 2× or 3× SDS-PAGE sample buffer (50 mM
Tris-HCl (pH 6.8), 100 mM DTT, 2% SDS, 10% glycerol, and
0.1% bromphenol blue).
Electrophoretic Analysis of Proteins--
Proteins or methylated
proteins were boiled in SDS-PAGE sample buffer at 100 °C for 5 min
(except where indicated) and separated on slab gels prepared from 29%
(w/v) acrylamide and 1% (w/v)
N,N-methylenebisacrylamide (1.5 mm × 5.5 cm-resolving gel) using the buffer system described by Laemmli (39) at
a constant current of 50 mA for ~2 h. Following electrophoresis, gels
were fixed with a fixing solution (45% (v/v) methanol and 10% (v/v)
acetic acid) and then soaked in Amplify (Amersham Pharmacia Biotech)
according to the manufacturer's instructions. Gels were dried in a
vacuum, and radioactivity was visualized by exposing the gels to an
x-ray film at Analysis of Methylated Arginines--
The enzyme assay was done
in a 500-µl reaction volume containing 80 µg of MBP, 10 µg of
purified FLAG-PRMT5, and 1 µCi of [14C]AdoMet. MBP was
then precipitated with 500 µl of 25% (w/v) trichloroacetic acid. The
precipitate was washed with acetone twice and then dissolved in
distilled water. The solution was dried in a 6 × 50-mm glass vial
and hydrolyzed with 6 N HCl at 110 °C for 24 h in a
Waters Pico-Tag work station (Pico-Tag® System, Waters,
Milford, MA). Released amino acids were labeled with
phenylisothiocyanate and separated on a Pico-Tag column according to
the recommended instructions of the manufacturer. Monomethylated and
symmetrically dimethylated arginines purchased from Sigma were also
labeled in the same manner and used as standards. The sample and the
standards were injected onto an HPLC column (Waters Pico-Tag, 3.9 × 300 mm) equilibrated with 140 mM sodium acetate buffer
containing 0.05% (v/v) triethylamine and 6% (v/v) acetonitrile at
46 °C, respectively. Amino acids were eluted from the column with
the acetonitrile gradient recommended by the manufacturer. Phenylisothiocyanate- and 14C-labeled amino acids were
simultaneously detected on a serially connected UV detector (269 nm)
and a Model 150TR on-line flow scintillation analyzer (Packard
Instrument Co.), respectively.
In Vivo Binding Assay--
To detect in vivo
interactions between PRMT5 proteins, 293T cells were transfected with
various sets of PRMT5 expression plasmids. After transfections, the
cells were broken in lysis buffer. Aliquots of cell lysates were
incubated with glutathione or anti-FLAG antibody beads for 2 h at
4 °C. The beads were then washed five times with lysis buffer. The
bead-bound proteins dissolved in 1× SDS-PAGE sample buffer with a
final concentration of 100 mM DTT were then boiled. Protein
samples were separated by SDS-PAGE and analyzed by Western blotting
with anti-GST or anti-FLAG antibodies.
Purification of FLAG-PRMT5 and FLAG-PRMT1 Proteins--
293T
cells (5 × 108) transfected with pCMV2-PRMT5 or
pCMV2-PRMT1 DNA were broken in lysis buffer as described above. The
cleared lysates were applied to an anti-FLAG affinity column (1 × 10 cm) equilibrated in lysis buffer, and the column was then washed
twice with lysis buffer. PRMT proteins were eluted with 100 mM glycine HCl buffer (pH 3.5). The protein elutes were
collected in 1 M Tris base buffer (pH 8.0) and then
dialyzed against PRMT buffer. The dialysates were concentrated using a
Nanospin Plus centrifugal filter (Gelman Instrument Co.).
Preparation of GST Fusion Proteins and Mouse Polyclonal Antibody
against PRMT5--
GST fusion proteins were expressed and purified as
described by the manufacturer (Amersham Pharmacia Biotech). In brief,
cells harboring GST fusion expression plasmids were induced with 1 mM isopropyl-1-thio-
A GST-PRMT5-C (amino acids 315-637) fusion protein was expressed as a
soluble form in E. coli. The fusion protein was purified as
described above. The purified fusion protein, which appeared to be
homogeneous on an SDS-polyacrylamide gel, was used to immunize BALB/c
mice for antibody production.
Sedimentation Analysis of PRMT5 and PRMT1 Proteins--
The
purified FLAG-PRMT5 or FLAG-PRMT1 protein (20-40 µg) was overlaid on
a 35-ml gradient of 5-45% sucrose in sedimentation buffer (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, and 1 mM phenylmethylsulfonyl
fluoride). After centrifugation at 10 °C for 24 h at 25,000 rpm
in a Beckman SW 28 rotor, the fractions were collected from the bottom.
To analyze a homo-oligomer of endogenous PRMT5 in human cells by
sedimentation, 293T cells (1 × 108) were washed once
with phosphate-buffered saline and resuspended in 3 ml of PRMT buffer.
The resuspension was sonicated for 30 s on ice. Cell debris was
removed by centrifugation. The soluble protein extract was sedimented
on a 35-ml 5-45% sucrose density gradient under the same conditions
as described above.
Chemical Cross-linking of PRMT5 Proteins by Glutaraldehyde
Treatment in Vitro--
Glutaraldehyde (40) was used to form chemical
bridges in the homo-oligomers of FLAG-PRMT5. The purified FLAG-PRMT5 or
FLAG-PRMT5-C protein (100 ng) was incubated with glutaraldehyde
(0.00006-0.0006%) in 20 µl of PRMT buffer for 5 min at room
temperature. The samples were boiled in SDS-PAGE sample buffer
containing 100 mM DTT for 5 min and then separated by 6%
SDS-PAGE. PRMT5 proteins were detected by Western blotting with an
anti-FLAG monoclonal antibody.
Subcellular Localization of Human PRMT5--
COS-1 or Chang's
liver cells (105 cells/ml) were cultured on a
polylysine-coated slide glass before transfection. As a control, pEGFP-PRMT5 or pEGFP-C1 (5 µg) was transfected. After 24 h, the cells were observed with a confocal microscope.
PRMT5 Contains the Conserved Sequence Motifs Present in a Family of
S-Adenosyl-L-methionine-dependent
Protein-arginine Methyltransferases--
Searches of the
GenBankTM/EBI Data Bank to identify the biochemical
function of PRMT5 revealed that full-size PRMT5 does not have
significant homology to any characterized proteins. We separated N- and
C-terminal regions of PRMT5 and searched to find whether the N- or
C-terminal region has amino acid sequence homology to characterized
proteins. The search revealed that whereas the N-terminal region does
not have any homology to the known genes, the C-terminal portion of
PRMT5 contains amino acid sequences homologous to domains I-III
present in AdoMet-utilizing methyltransferases including PRMT1 (9,
41-43) (Fig. 1). The amino acid sequence
of the post-I domain of PRMT5 (Lys-Tyr-Ala-Val-Glu) matches well with
that of human PRMT2 (Val-Tyr-Ala-Val-Glu) (19, 20). Furthermore,
intervals between the conserved domains in PRMT5, PRMT1, and PRMT3 are
also well preserved. These observations led us to examine the
possibility of whether PRMT5 contains intrinsic protein-arginine
methyltransferase activity.
PRMT5 Contains Intrinsic Protein-arginine Methyltransferase
Activity--
The GST-PRMT5 plasmid, with GST at the N terminus
of the gene for PRMT5, and a control plasmid (GST) were
transfected into 293T cells, respectively. The cell lysates were
pulled-down with glutathione-agarose beads. The bead suspensions were
incubated with [14C]AdoMet and MBP. The reaction products
were visualized by SDS-PAGE and fluorography (Fig.
2A). MBP was radioactively
labeled only by the beads incubated with the cell lysate transfected
with the GST-PRMT5 plasmid, but not by the beads from the control
plasmid. The GST-PRMT5 protein bound to the beads was detected by
Western blotting with anti-GST antibody (Fig. 2A). To
further demonstrate the protein methyltransferase activity of PRMT5,
the FLAG-PRMT5 protein was partially purified from the 293T cell lysate
transfected with the FLAG-PRMT5 plasmid via anti-FLAG
antibody-conjugated agarose beads. Two closely migrating protein bands
of ~72 kDa in size in the enzyme preparation were detected by Western
blotting with anti-PRMT5-C antibody and Coomassie Blue staining (Fig.
2B). The fast migrating band of 72 kDa was not detected by
Western blotting with anti-FLAG antibody (data not shown), suggesting that this protein is endogenous PRMT5 and that a homomeric complex between FLAG-PRMT5 and endogenous PRMT5 is formed. MBP was methylated by purified FLAG-PRMT5 in a dose-dependent manner (Fig.
2B, 14C-Methylation). Next, we determined
whether PRMT5 methylates the arginine residue in MBP. The acid lysate
of MPB methylated by FLAG-PRMT5 was subjected to separation by HPLC as
described under "Experimental Procedures." 14C-Labeled
monomethylarginine and dimethylarginine comigrated with the standards
monomethylarginine and symmetric dimethylarginine (Fig.
2C). To further demonstrate that the catalytic activity of
PRMT5 is intrinsic and not derived from a contaminant from human cells
during purification, we examined the enzyme activity of GST-PRMT5
purified from E. coli. It was reported that E. coli does not contain any protein-arginine methyltransferase (3). Furthermore, the conserved GXGXG motif (domain I)
in the AdoMet-dependent protein methyltransferases is known
to be an AdoMet-binding site and is critical for the catalytic activity
(41-43). Therefore, we constructed a domain I mutant in which the
amino acids in the GAGRG motif of PRMT5 are substituted with RARLE. The
GST-PRMT5 protein expressed in E. coli methylated MBP,
whereas the domain I mutant (GST-PRMT5-M) did not (Fig.
3A). Taken together, the results indicate that PRMT5 contains intrinsic protein-arginine methyltransferase activity.
The C-terminal Region of PRMT5 Containing All the Methyltransferase
Motifs Is Not Sufficient for the Catalytic Activity and Interacts with
the N-terminal Domain of PRMT5--
The domains for the catalytic
activity are localized in the C terminus of PRMT5 from amino acids 359 to 637 (Fig. 1). To determine whether only the C-terminal domain of
PRMT5 containing canonical methyltransferase motifs is sufficient for
the enzyme activity, the purified FLAG-PRMT5-C-(308-637) and
GST-PRMT5-C-(315-637) proteins bound to glutathione-agarose beads were
used in the enzyme reactions using MBP and histone as substrates,
respectively. Neither the FLAG-PRMT5-C-(308-637) nor
GST-PRMT5-C-(315-637) protein showed the enzyme activity, whereas the
full-size FLAG-PRMT5 and GST-PRMT5 proteins did (Fig. 3, B
and C). The GST-PRMT5-C-(315-637) protein expressed in
E. coli also did not show the enzyme activity (Fig. 3A). The results suggest that the C-terminal domain of PRMT5
by itself is not sufficient for the catalytic activity.
It was suggested that Skb1, a yeast homolog of human PRMT5, forms a
homomeric complex in fission yeast and that the N terminus of Skb1 may
be important for the homomeric complex formation (24). Furthermore,
purified FLAG-PRMT5 appeared to form a homomeric complex (Fig.
2B). Therefore, we examined whether human PRMT5 forms a
homomeric complex in vivo by co-immunoprecipitation.
FLAG-PRMT5 and glutathione-PRMT5 plasmids were cotransfected into 293T
cells. The cell lysates were precipitated with anti-FLAG antibody- or GST-conjugated agarose beads. The immunoprecipitates or the
GST-bead-bound proteins were separated by SDS-PAGE and analyzed by
immunoblotting using anti-GST or anti-FLAG antibodies (Fig.
4). GST-PRMT5 was found to be associated
with FLAG-PRMT5 and vice versa. To determine whether an interaction
between N- and C-terminal domains occurs, we cotransfected the
FLAG-PRMT5-C-(308-637) and GST-PRMT5-N- (1) plasmids into 293T
cells. FLAG-PRMT5-C-(308-637) was coprecipitated with
GST-PRMT5-N-(1-309) and vice versa (Fig. 4). The results indicate that
PRMT5 forms a homomeric complex in vivo and suggest that an
interaction between the N- and C-terminal regions of PRMT5 is involved
in the homomeric complex formation.
PRMT5 Forms Homomeric Complexes, Including a Dimer and
Tetramer--
The homomeric complex of PRMT5 was further characterized
by sedimentation analysis on a sucrose gradient. As PRMT1 was shown to
form an oligomeric complex in Rat1 cells (9, 10), we also characterized
the oligomeric complex of purified PRMT1. The purified FLAG-PRMT5 and FLAG-PRMT1 proteins were sedimented onto 35-ml 5-45%
sucrose gradients. The fractions were collected from the bottom of the
gradients. Equal volumes of every other gradient fraction boiled in
sample buffer containing a reducing agent were separated by SDS-PAGE
and subjected to Western blot analyses with anti-FLAG antibody (Fig.
5A). The enzyme assay was
carried out with equal volumes of the gradient fractions and MBP as a
substrate. The radioactivity incorporated into MBP was quantified by a
PhosphorImager (Fig. 5B). The FLAG-PRMT5 protein peaked
mainly at two positions. The first position at which it peaked is
fraction 41, which sedimented between standard molecular masses of 68 and 158 kDa. Most likely, this fraction represents a homodimer,
although we cannot rule out that it may partially contain a monomer.
The second position at which it peaked is fraction 25, which
corresponds to a molecular mass of slightly >240 kDa. This fraction
may represent multimers, including a tetramer. The enzyme activity
comigrated with the dimer and multimer fractions of FLAG-PRMT5. Most of
FLAG-PRMT1 sedimented between standard molecular masses of 158 and 240 kDa, suggesting the presence of a multimer, including a tetramer (Fig. 5A). A minor portion of FLAG-PRMT1 sedimented at a standard
molecular mass of 68 kDa, suggesting the presence of a dimer. The
MBP-methylating activity of FLAG-PRMT1 was mainly detected in the
multimer fractions (Fig. 5B).
The presence of a dimer and multimers of purified FLAG-PRMT5 was
further demonstrated by a chemical cross-linking experiment. Purified
FLAG-PRMT5 or FLAG-PRMT5-C-(308-637) was treated with glutaraldehyde,
which forms intra- or intercovalent chemical bridges between proteins
depending on its concentration (40). The reaction products boiled in
sample buffer containing a reducing agent were separated by SDS-PAGE
and analyzed by Western blotting with anti-FLAG antibody (Fig.
6A). Three protein bands,
namely a monomer of 72 kDa, a homodimer migrating between standard
molecular mass markers of 200 and 97.4 kDa, and a multimer >200 kDa in
size, were detected in the FLAG-PRMT5 sample. FLAG-PRMT5-C-(308-637)
did not form oligomeric complexes. Taken together, the results indicate
that PRMT5 forms homo-oligomers, including a dimer and tetramer.
Both Covalent Linkage via Disulfide Bond and Noncovalent
Association Are Involved in the Formation of Homo-oligomers of PRMT5
and PRMT1--
We examined whether the homo-oligomers of FLAG-PRMT5
and FLAG-PRMT1 are linked covalently by disulfide bond or by
noncovalent association. Purified FLAG-PRMT5 boiled in SDS-PAGE sample
buffer with or without a reducing agent was separated by SDS-PAGE. The PRMT5 protein was detected by Western blotting with anti-FLAG antibody
(Fig. 6B, Pooled). Only a monomer was detected in
the presence of 100 mM DTT, whereas a monomer and smeared
bands containing presumably a dimer and multimer of FLAG-PRMT5 were
detected in the absence of DTT, indicating that a portion of PRMT5
homo-oligomers are covalently linked by a disulfide bond. The presence
of a monomer in the absence of a reducing agent suggests either that a
monomer is present in purified FLAG-PRMT5 at a small quantity or that the oligomeric complexes are formed partially by noncovalent
interaction of a monomer. To further determine the presence of covalent
and noncovalent associations in the oligomeric complexes, we used the
dimer and multimer fractions of FLAG-PRMT5, which were separated by
sedimentation (fractions 25 and 41) (Fig. 5). The dimer and multimer
fractions boiled in SDS-PAGE sample buffer with or without a reducing
agent or in low concentrations of a reducing agent were separated by
SDS-PAGE. Protein bands were detected by Western blotting with
anti-FLAG antibody (Fig. 6B). In the presence of 10 and 100 mM DTT, the dimer fraction was dissociated completely into
a monomer, whereas in the absence or at low concentrations of DTT, both
the monomer and dimer were detected, indicating that a portion of the
dimer is covalently associated by disulfide bond (fraction 41). The
multimer fraction was also completely dissociated into a monomer in the
presence of 10 and 100 mM DTT, whereas the monomer, dimer,
and multimer were detected in the absence or at low concentrations of
DTT (fraction 25). The multimer fraction apparently did not contain
FLAG-PRMT5 present as a monomer, at least in the sedimentation
analysis. The presence of a monomer and dimer in the absence or at low
concentrations of DTT indicates that a portion of the multimer is
formed by noncovalent interaction of a monomer or a disulfide-linked
dimer and is SDS-labile. Furthermore, the presence of a multimer in the
absence of DTT indicates that a portion of the multimer is formed by
covalent association by disulfide bonds and is SDS-resistant. Thus, the
results indicate that a PRMT5 multimer can be formed either by covalent
association by disulfide bond or by noncovalent association of a
monomer or dimer linked by a disulfide bond. A multimer can also be
heterogeneous depending on the association properties. A monomer and
dimer in the multimer fraction would not have been detected in the
absence of a reducing agent if a noncovalent association were not
involved in the homo-oligomerization.
Multimer fraction 33 of FLAG-PRMT1 separated by sedimentation (Fig. 5)
was dissociated into a monomer in the presence of 10 and 100 mM DTT, indicating that covalent linkage by a disulfide bond is involved in the homo-oligomerization (Fig. 6B,
Fraction #33). A monomer and dimer, as well as a smeared
band >97.4 kDa in size, were detected in the absence or at low
concentrations of the reducing agent, indicating that homo-oligomeric
complexes of PRMT1 are also formed by noncovalent association of a
monomer or a covalently linked dimer.
PRMT5 Is Present as a Homo-oligomer in Vivo--
To determine
whether endogenous PRMT5 is also present in oligomeric forms and as a
predominant enzyme methylating MBP in cells, we sedimented the protein
extract of 293T cells on a sucrose gradient. Equal volumes of the
gradient fractions were separated by SDS-PAGE. An endogenous PRMT5
protein was then detected by Western blotting with anti-PRMT5-C
antibody (Fig. 7A). The enzyme
activities in the sedimentation fractions were measured using MBP as a
substrate. 14C-Labeled proteins were visualized by SDS-PAGE
and fluorography (Fig. 7C). The radioactivity incorporated
into MBP was quantified by a PhosphorImager (Fig. 7B). The
endogenous PRMT5 protein peaked at fraction 17, which appears to
correspond to the molecular mass of the tetramer of PRMT5 (4 × 72 = 288 kDa). Monomeric and dimeric forms of endogenous PRMT5
were not detected. The MBP-methylating enzyme activity was detected in
a broad range of fractions containing the PRMT5 protein, and it
appeared to peak at the fractions in which PRMT5 was not detected,
suggesting that PRMT5 is not predominant (Fig. 7B). However,
we cannot completely exclude that PRMT5 is a predominant
MBP-methylating enzyme because endogenous proteins methylated by
cellular enzymes (Fig. 7C, arrowheads) interfered with specific detection of 14C-labeled MBP.
PRMT5 Contains Lower Specific Enzyme Activity Compared with PRMT1
and Differs in Cellular Localization from PRMT1--
To compare the
specific enzyme activity of PRMT5 with that of PRMT1, we used a GST-GAR
(glycine/arginine-rich) protein
expressed in E. coli as a substrate in the enzyme assay.
GST-GAR is a recombinant protein containing the first 148 amino acids
of the human fibrillarin protein fused in frame to GST. The
amino-terminal region of fibrillarin contains 14 arginine residues, the
majority of which are present in RGG consensus methylation sites
(44-46). The enzyme assay was carried out at different molar
concentrations of purified FLAG-PRMT1 and FLAG-PRMT5 proteins. The
reaction products were visualized by SDS-PAGE and fluorography, and
then they were quantified by a PhosphorImager (Fig.
8). PRMT5 contained ~2.5-fold weaker
specific enzyme activity compared with PRMT1.
PRMT1 is localized in the nucleus of Rat1 cells, whereas PRMT3 is
predominantly cytoplasmic (10). We examined where human PRMT5 is
localized in the cell. We fused the gene for GFP in frame to the N
terminus of PRMT5. The GFP-PRMT5 plasmid was transfected into COS-1 and
Chang liver cells, and then we examined green fluorescence under
fluorescence microscopy (Fig. 9). Green
fluorescence was detected mainly at in the cytoplasm of COS-1 and Chang
liver cells, suggesting that PRMT5 is predominantly localized in the
cytoplasm.
We demonstrated here that PRMT5 (Skb1Hs/JBP1) contains an
intrinsic protein-arginine methyltransferase activity. First,
the conclusion that the catalytic activity of PRMT5 is intrinsic is supported by the facts that primarily the conserved motifs of the known
AdoMet-dependent protein-arginine methyltransferases are
present in PRMT5 and that spacings between the motifs are not greatly
different from those of other PRMTs (Fig. 1). Second, monomethyl- and
dimethylarginines were detected in the acid lysate of MBP methylated by
PRMT5 (Fig. 2C). Third, GST-PRMT5 expressed in E. coli, but not GST-PRMT5 domain I mutant, contained the enzyme activity (Fig. 3A). Finally, the PRMT activities comigrated
with the oligomeric complexes of purified FLAG-PRMT5 sedimented on a
sucrose gradient (Fig. 5).
The presence of a homomeric complex of PRMT5 in vivo was
detected by co-immunoprecipitation (Fig. 4). We provide biochemical evidence that the homomeric complex of FLAG-PRMT5 is composed of a
dimer and multimer by sedimentation analysis, chemical cross-linking, and SDS-PAGE in the presence or absence of a reducing agent (Figs. 5
and 6). The oligomerization was not due to a FLAG epitope tagged at the
N terminus of PRMT5 because FLAG-PRMT5-C-(308-637) did not form a
homo-oligomer (Fig. 6A). Although the presence of the endogenous PRMT5 multimer in vivo was detected in the
protein extract of 293T cells by sedimentation, an endogenous PRMT5
dimer was not detected (Fig. 7). It is likely that cells expressing FLAG-PRMT5 overproduce dimers simply as an intermediate for the formation of multimers. Alternatively, a FLAG epitope tagged at the N
terminus of PRMT5 may partially interfere with efficient formation of
homo-oligomers larger than dimers. The multimer might be composed of a
tetramer, a hexamer, an octamer, and so on. However, sedimentation
analyses suggested that endogenous PRMT5 or FLAG-PRMT5 is present most
likely as a tetramer (Figs. 5 and 7), although we cannot rule out the
presence of a multimer larger than a tetramer in size in
vivo, which is likely unstable during purification or
sedimentation analysis. PRMT1 also appears to be present as multimers,
including a tetramer, because it migrates between standard molecular
masses of 158 and 240 kDa (Fig. 5). In fact, Lin et al. (9)
reported that endogenous PRMT1 migrated as a high molecular mass
complex of ~180 kDa on a Superdex 200 gel filtration column, although
it eluted in a broad peak ranging between 200 and 440 kDa on a
Sephacryl S300HR gel filtration column (10).
The specific enzyme activity of GST-PRMT5 from E. coli was
several hundredfold lower than that of GST-PRMT5 from mammalian cells
(Fig. 3A), presumably because GST-PRMT5 expressed in
mammalian cells is likely to form a homomeric complex with endogenous
PRMT5. GST-PRMT5 from E. coli appeared not to form distinct
homomeric complexes (data not shown). The C-terminal portion of PRMT5,
which contains three important motifs for the PRMT activity, showed by
itself neither the enzyme activity nor oligomerization (Figs. 3 and
6A). An interaction between the N- and C-terminal domains of
the PRMT5 protein, which was detected by co-immunoprecipitation, appears to be involved in oligomerization of PRMT5 (Fig. 4). The multimer fractions of FLAG-PRMT5 contained higher specific enzyme activity compared with the dimer fractions, given that the dimer fractions contained much more PRMT5 protein than the multimer fractions
as determined by Western blot analysis (Fig. 5). These results suggest
that homo-oligomerization of PRMT5 is important for the catalytic activity.
The gene for type II PRMT, which produces symmetric
dimethylarginine, has not been cloned. PRMT5 methylated MBP,
histone, and GST-GAR as a designed methyl acceptor (Figs. 2, 3, and 8). Histones and GST-GAR have been shown to be efficient methyl acceptors for PRMT1, PRMT3, and yeast Rmt1 (arginine
methyltransferase-1) (9, 10, 47),
which represent type I enzymes. Previous studies on the mammalian PRMTs
showed that the enzyme responsible for methylating MBP is distinctly
different from the histone-methylating enzyme (48). The MBP-specific
methyltransferase preferentially methylates MBP and histone to a much
lesser extent. The histone-specific enzyme methylates only histone. We
could not determine conclusively whether dimethylarginine detected in
the acid lysate of MBP methylated by PRMT5 is symmetric or asymmetric
and, consequently, whether PRMT5 is a type I or II enzyme. The HPLC
column used for the detection of methylated arginines could not readily
resolve symmetric and asymmetric dimethylarginines, although
dimethylarginine in the acid lysate of MBP methylated by PRMT5
comigrated with a standard symmetric dimethylarginine (Fig.
2C). As PRMT5 methylates MBP and histone, PRMT5 may be an
enzyme known as an MBP-specific methyltransferase.
Immunoaffinity-purified FLAG-PRMT1 methylated MBP (Fig. 5). However, it
was reported that rat PRMT1 and Rmt1, a yeast homolog of human PRMT1,
did not methylate MBP in vitro (9, 47). Purified FLAG-PRMT1
was not contaminated with PRMT5 since the FLAG-PRMT1 enzyme preparation
did not contain PRMT5 as determined by Western blot analysis and
FLAG-PRMT1 did not interact with PRMT5 as determined by
co-immunoprecipitation analysis (data not shown). The discrepancy from
the previous results may result from MBP derived from different sources. We used MBP derived from guinea pig brain (Figs. 2, 3, and 5),
whereas bovine brain MBP was used as a methyl acceptor in the cases of
rat GST-PRMT1 and yeast GST-Rmt1 proteins expressed in E. coli (9, 47). However, MBP derived from bovine brain was
methylated by PRMT5 (JBP1) (23). These findings make it highly unlikely
that MBP from different sources determines whether or not it functions
as a methyl acceptor for protein-arginine methyltransferases. On the
contrary, in vitro substrate specificity may depend on an
enzyme preparation. FLAG-PRMT1 purified from mammalian cells may have
different substrate specificity compared with recombinant GST-PRMT1
expressed in E. coli. This may account for the
MBP-methylating activity of FLAG-PRMT1.
Presumably, the substrate and reaction product specificity of
protein-arginine methyltransferases may be partially dependent on the
heterogeneity of homo-oligomeric complexes. This is presumed since
purified PRMT1 and PRMT5 preparations are composed of dimers and
multimers, which can be separated by sucrose gradient sedimentation (Fig. 5). Furthermore, PRMT1 and PRMT5 multimers are likely to be
composed of at least three different kinds of multimers formed by
noncovalent association of a monomer or a disulfide-linked dimer or by
covalent association via disulfide linkage (Fig. 6B). It is
possible that noncovalently associated, large homo-oligomeric complexes
of native PRMTs present in vivo might be dissociated into a
more stable homo-oligomeric complex during purification by
chromatographic methods from cells or tissues. This dissociation would
result in different substrate and reaction product specificity. For
example, although the spliceosomal D1 protein in HeLa cells is found to
be symmetrically dimethylated in vivo, the D1 protein overexpressed in baculovirus-infected insect cells is asymmetrically arginine-methylated. A heptapeptide (GRGRGRG) also present in the D1
protein is asymmetrically methylated by the type II MBP-specific methyltransferase in vitro (8). Therefore, the findings
suggest that the tenuous classification of type I and II PRMTs based on substrate and reaction product specificity in vitro should
be reconsidered.
A heterogeneity of the homo-oligomeric complexes of PRMTs resulting
from the association characteristics (Figs. 5 and 6) and the
presence of various protein-arginine methyltransferases methylating MBP
and histones in cells (1, 22, 23, 47) might have also resulted in
multiple protein bands in the highly purified PRMT enzyme preparation.
In fact, although a number of purifications of protein-arginine
methyltransferase have been reported (3, 36, 37), the polypeptide
composition of protein-arginine methyltransferase has been
controversial. For example, histone-arginine methyltransferase from
calf brain contains two polypeptides of 110 and 75 kDa (36), whereas
Rawal et al. (37) showed that the enzyme from rat liver contains only a single 110-kDa polypeptide. The purest protein-arginine methyltransferase fraction from HeLa cells contains about eight protein
bands upon SDS-PAGE and silver staining, and the two most prominent
bands have molecular masses of 45 and 100 kDa (3). Presumably, the two
bands may represent a monomer and disulfide-linked dimer of PRMT1. Our
result that the disulfide-linked dimer of PRMT1 is not completely
dissociated into a monomer even in the presence of 1 mM DTT
(Fig. 6B, Fraction #33) supports this possibility.
PRMT5 has been shown to bind the pICln (I = current,
Cl = chloride, and n = nucleotide-sensitive)
protein, which is correlated with the appearance of a
nucleotide-sensitive chloride current (49). The pICln protein was shown
previously to exist in several discrete complexes with other cytosolic
proteins (50). PRMT5 binds to Janus kinases, which are localized in the
cytoplasm and are involved in an interferon-signaling pathway (23,
51-53). We found PRMT5 as a molecule interacting with the NS3 protein of HCV, which is also localized in the cytoplasm (54). The
sedimentation profile of purified PRMT1 and PRMT5 indicates that they
are distinctly different enzymes (Fig. 5). Thus, the results suggest
that although PRMT1 functions mainly in the nucleus (10), PRMT5 plays
important roles in the cytoplasm.
The HCV NS3 protein is a multifunctional protein containing serine
protease and RNA helicase activities (28-33). A specific interaction
between the C-terminal domain of PRMT5 and the N-terminal domain of NS3
was detected in vitro and in
vivo.2 However, PRMT5
appeared not to affect the enzyme activities of HCV NS3. A reason,
then, why PRMT5 interacts with NS3 is that HCV NS3 is
post-translationally arginine-methylated by a protein-arginine methyltransferase, and it may possibly modulate the enzyme activity in
HCV-infected cells. In fact, we found that HCV NS3 as an RNA-binding protein contains several Arg-Gly motifs and that arginine residues in
the NS3 helicase domain are methylated by cellular protein-arginine methyltransferase.2 Therefore, it will be of great interest
to determine the functions of PRMT5 interacting with the HCV NS3
protein in mammalian cells and the biological consequences of protein
arginine methylation. The findings that PRMT5 and PRMT1 homo-oligomers
formed by covalent and noncovalent interactions can be heterogeneous
may contribute to understanding the polypeptide compositions of
protein-arginine methyltransferases and identifying their specific
substrates and reaction products.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
receptor (18). The gene for PRMT2 was
found by screening expressed sequence tag data bases (19, 20), but its
protein methyltransferase activity has not been detected. PRMT3
containing a zinc finger domain in its amino terminus (21) was found by
conducting a yeast two-hybrid screen using rat PRMT1 as a bait (10).
CARM1/PRMT4 regulates transcription as an interacting molecule with
GRIP1 (glucocorticoid
receptor-interacting
protein-1), a p160 family of transcriptional
coactivators (22). Pollack et al. (23) found that Skb1Hs
(24, 25), named JBP1 (Janus kinase-binding protein-1), interacts with Janus kinases and
contains the protein methyltransferase activity. However, it was not
clearly determined whether the protein methylation activity of JBP1 is
arginine-specific. Any biological consequences of an interaction
between Janus kinases and JBP1 are unknown.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C for 3-14 days or by a Fuji BAS1000
PhosphorImager. 14C incorporation was quantitated using a
PhosphorImager. Gels were stained with Coomassie Brilliant Blue R-250
for 20-30 min and destained with the fixing solution to visualize the
protein bands. Proteins on the gels were transferred to polyvinylidene
difluoride membranes (PerkinElmer Life Sciences). Membranes were
blocked with Tris-buffered saline (50 mM Tris-HCl (pH 7.4)
and 150 mM NaCl) containing 5% skim milk and then
incubated with mouse anti-FLAG or anti-GST antibodies at a
concentration of 5 µg/ml. Membranes were washed three times with
Tris-buffered saline and incubated with goat anti-mouse IgG conjugated
to horseradish peroxidase (Sigma) at 1:5000 dilution. After being
washed three times, the reactive proteins were detected by enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech).
-D-galactopyranoside for
2-3 h at 30 °C. Cells were washed with phosphate-buffered saline
buffer, resuspended in lysis buffer, and then sonicated. Soluble
protein extracts were loaded onto glutathione-agarose columns (1 × 10 cm). The columns were washed four times with lysis buffer.
Bead-bound proteins were eluted with PRMT buffer containing 10 mM reduced glutathione. After dialysis, purified proteins
were stored at
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (90K):
[in a new window]
Fig. 1.
Human PRMT5 contains conserved domains
of AdoMet-dependent protein-arginine
methyltransferases. The amino acid sequence of human PRMT5
(hPRMT5) was deduced from human PRMT5 cDNA sequenced in
our laboratory. To clone a murine homolog of human PRMT5, primers
5'-GTCCTCCACGTAATGCCTATGACC-3', 5'-CTCTAGCGTCACCACGGCATT-3',
5'-GTCCTCCACCTAATGCCTATGACC-3', and 5'-CAAGGCTCTGGACACTTGGC-3' were
used in the polymerase chain reactions to amplify a partial fragment of
PRMT5 from a mouse T-cell hybridoma cDNA library. The partial
murine PRMT5 (mPRMT5) clones were sequenced. The amino acid
sequence of murine PRMT5 missing perhaps six amino acids at its N
terminus was deduced from the partial murine PRMT5 cDNA
clones and GenBankTM/EBI accession numbers
AA120484, AA066095, AA838892, AA266241, AA921641, AA560154, AA270442,
AA755892, AA637429, AA139488, AA869821, AA967893, and AA638905. Murine
PRMT5 is almost identical to human PRMT5 at the amino acid level. The
amino acid sequences of human PRMT1 and PRMT3 were deduced from
GenBankTM/EBI accession numbers AA307385 and
Q99873. Conserved domains I, post-I, II, and III of protein-arginine
methyltransferases are indicated by lines. Identical amino
acid residues are indicated in boldface.
View larger version (39K):
[in a new window]
Fig. 2.
Human PRMT5 contains
AdoMet-dependent protein-arginine methyltransferase
activity. A, GST-PRMT5 contains MBP-specific
methyltransferase activity. An expression vector encoding GST (pEBG) or
GST-PRMT5 (pEBG-PRMT5) was transfected into 293T cells. After 36 h, the cells were extracted in 1 ml of lysis buffer as described under
"Experimental Procedures." Soluble protein extracts were incubated
with glutathione-agarose beads. After being washed, the beads were
incubated in 30 µl of PRMT buffer containing 5 µg of MBP (guinea
pig brain; Sigma) and 0.25 µCi of [14C]AdoMet for
2 h at 30 °C. The reaction products dissolved in sample buffer
containing 100 mM DTT were boiled and separated on two
polyacrylamide gels by SDS-PAGE. A gel was fixed and treated with
Amplify for 30 min, dried, and visualized using a PhosphorImager
(upper panel). The other gel was stained with Coomassie Blue
to visualize MBP (middle panel). To detect GST-PRMT5 bound
to the beads, the bead-bound proteins were dissolved in sample buffer
containing 100 mM DTT and boiled. The supernatants were
analyzed by SDS-PAGE and Western blotting with anti-GST antibody
( -GST) (lower panel). The amounts
of transfected DNAs and molecular mass markers (in kilodaltons) are
shown at the top and left, respectively. B,
immunoaffinity-purified FLAG-PRMT5 contains MBP-specific
methyltransferase activity. A FLAG-tagged expression vector encoding
PRMT5 (FLAG-PRMT5) was transfected into the 293T cells (5 × 108). The FLAG-PRMT5 protein was purified from the
transfected cell lysate on anti-FLAG antibody-conjugated agarose beads
(FLAG M2-agarose, Sigma) as described under "Experimental
Procedures." The purified protein was dissolved in sample buffer
containing 100 mM DTT as the final concentration and then
boiled. The protein sample was separated on two polyacrylamide gels by
SDS-PAGE. A gel was analyzed by Western blotting with mouse
anti-PRMT5-C antibody in 1:2000 dilution. The other was stained with
Coomassie Blue. The protein band marked as PRMT5 represents
endogenous PRMT5. In vitro protein methylation reactions
were performed as a function of FLAG-PRMT5 concentration (0-200 ng) in
40-µl reaction volumes containing 5 µg of MBP and 0.25 µCi of
[14C]AdoMet. The reaction products were visualized by
SDS-PAGE and fluorography (14C-Methylation).
M, molecular mass markers. C, identification of
the 14C-methylated arginine residues in MBP methylated by
PRMT5. MBP was methylated by purified FLAG-PRMT5 in the presence of
[14C]AdoMet as described under "Experimental
Procedures." The presence of
NG-[methyl-14C]monomethylarginine
(MMA) and symmetric
NG,N'G-[methyl-14C]dimethylarginine
(DMA) (lower line) was observed by separating the
hydrolyzed amino acids on a reverse-phase HPLC column using unlabeled
monomethylarginine and symmetric dimethylarginine as standards
(upper line).
View larger version (48K):
[in a new window]
Fig. 3.
A, the GST-PRMT5 fusion protein
expressed in E. coli contains MBP-specific methyltransferase
activity. GST-PRMT5 fusion proteins purified from 293T cells and
E. coli were used in the enzyme assay. In vitro
methylation reactions were performed with GST (30 µg), GST-PRMT5
(0.25-30 µg), GST-PRMT5-C (30 µg), or GST-PRMT5-M (30 µg); 5 µg of MBP; and 0.25 µCi of [14C]AdoMet. The reaction
products were fluorographed (14C-Methylation) as
described in the Fig. 1 legend. B, the C-terminal domain of
FLAG-PRMT5 by itself doses not methylate MBP. The FLAG-PRMT5-C protein
was purified from 293T cells transfected with the
FLAG-PRMT5-C-(308-637) plasmid by anti-FLAG antibody immunoaffinity
chromatography. The purified FLAG-PRMT5 or FLAG-PRMT5-C protein (100 ng) and MBP (5 µg) were used in the enzyme reaction. Reaction
products and PRMT5 proteins dissolved in sample buffer containing 100 mM DTT as a final concentration were boiled and separated
by SDS-PAGE. The methylation product was detected by fluorography
(14C-Methylation). PRMT5 proteins were detected by
Western blotting with anti-FLAG antibody
( -FLAG). C, GST-PRMT5, but not
GST-PRMT5-C, methylates histone. The GST-PRMT5-C-(315-637),
GST-PRMT5-N-(1-309), or GST-PRMT5 plasmid was transfected into 293T
cells. The expressed proteins were pulled-down from the cell lysates by
glutathione-agarose beads. In vitro methylation reactions
were performed with GST-PRMT5 proteins bound to the beads, 5 µg of
histone (type II-AS from calf thymus, Sigma), and
[14C]AdoMet. Reaction products and proteins bound to the
beads were dissolved in sample buffer containing 100 mM DTT
as a final concentration and separated by SDS-PAGE. Methylated proteins
were detected by fluorography (14C-Methylation).
PRMT5 proteins were detected by Western blotting with anti-GST antibody
(
-GST).
View larger version (40K):
[in a new window]
Fig. 4.
PRMT5 forms a homomeric complex in
vivo, and the N terminus of PRMT5 interacts with the C
terminus of PRMT5. The FLAG-PRMT5/GST-PRMT5 and
FLAG-PRMT5-N/GST-PRMT5-C plasmids or the GST (pEBG)-PRMT5/FLAG-PRMT5
plasmids as controls were cotransfected into 293T cells. Cell lysates
were prepared 36 h post-transfection. The cleared cell lysates
were incubated with anti-FLAG antibody- or glutathione-conjugated
beads. The beads were then treated as described under "Experimental
Procedures." A, proteins coprecipitated with GST-PRMT5
fusion proteins were dissolved in sample buffer containing 100 mM DTT as the final concentration, and they were then
detected by SDS-PAGE and Western blotting with anti-FLAG antibody
( -FLAG; right panel). GST-PRMT5
fusion proteins bound to the beads were detected by Western blotting
with anti-GST antibody (
-GST; left
panel). B, proteins coprecipitated with FLAG-PRMT5
proteins were dissolved in sample buffer containing 100 mM
DTT as the final concentration, and they were then detected by SDS-PAGE
and Western blotting with anti-GST antibody (right panel).
FLAG-PRMT5 proteins bound to the beads were detected by Western
blotting with anti-FLAG antibody (left panel). The GST-PRMT5
and FLAG-PRMT5 proteins are indicated by arrows. Molecular
mass markers are indicated. IP, immunoprecipitation.
View larger version (43K):
[in a new window]
Fig. 5.
Sedimentation analyses of the purified
FLAG-PRMT5 and FLAG-PRMT1 proteins. 293T cells (5 × 108) were transfected with the FLAG-PRMT5 or FLAG-PRMT1
expression plasmid. The FLAG-PRMT5 or FLAG-PRMT1 protein was purified
by anti-FLAG antibody-conjugated agarose beads. The purified proteins
were sedimented through a 35-ml 5-45% sucrose gradient. A,
equal volumes of every other gradient fraction were dissolved in sample
buffer containing 100 mM DTT as a final concentration and
separated by SDS-PAGE. The gels were subjected to Western blotting with
anti-FLAG antibody ( -FLAG) to detect the PRMT5
and PRMT1 proteins. B, the MBP-specific methyltransferase
assay was carried out with equal volumes of the gradient fractions in
the presence of [14C]AdoMet. Methylation products were
analyzed by SDS-PAGE and fluorography. 14C incorporation
was quantified using a PhosphorImager. The dimer (Di) and
multimer (Mu) positions are indicated by
arrowheads in A. Molecular mass markers
(Combitek, Roche Molecular Biochemicals) sedimented in a parallel
sucrose gradient are indicated by arrowheads in
B.
View larger version (32K):
[in a new window]
Fig. 6.
The homo-oligomeric complexes of FLAG-PRMT5
and FLAG-PRMT1 contain dimers and multimers formed by covalent linkage
via disulfide bonds and noncovalent interaction. A,
purified FLAG-PRMT5 contains a homodimer and multimer. The purified
FLAG-PRMT5 and FLAG-PRMT5-C proteins (200 ng) were incubated in 40 µl
of PRMT buffer with or without 0.00006 or 0.0006% glutaraldehyde
(GA) at room temperature for 5 min. Reaction products were
dissolved in SDS-PAGE sample buffer containing 100 mM DTT
as a final concentration and boiled. The FLAG-PRMT5 (left
panel) and FLAG-PRMT5-C (right panel) samples were
separated by 6% SDS-PAGE. PRMT5 proteins were detected by Western
blotting with anti-FLAG antibody ( -FLAG).
B, homo-oligomeric complexes of PRMT5 and PRMT1 are formed
by covalent linkage via disulfide bonds and noncovalent association.
Purified FLAG-PRMT5 (200 ng; Pooled) was boiled in SDS-PAGE
sample buffer with or without 100 mM DTT as the final
concentration for 5 min. Portions of fractions 41 and 25 of FLAG-PRMT5
and fraction 33 of FLAG-PRMT1 in Fig. 5 were boiled in SDS-PAGE sample
buffer with 0.1-100 mM DTT as the final concentration or
without DTT. The protein samples were then separated by 6% SDS-PAGE.
The PRMT5 and PRMT1 proteins were detected by Western blotting with
anti-FLAG antibody. Molecular mass markers are indicated. Monomer
(Mo), dimer (Di), and multimer (Mu)
positions are indicated by arrows.
View larger version (46K):
[in a new window]
Fig. 7.
Sedimentation analysis of crude extract of
293T cells. The soluble protein extract from 293T cells (1 × 108) was sedimented through a 35-ml 5-45% sucrose
gradient. Equal volumes of every other gradient fraction were boiled in
SDS-PAGE sample buffer containing 100 mM DTT as the final
concentration for 5 min. The protein samples were analyzed by SDS-PAGE
and Western blotting with anti-PRMT5-C antibody
( -PRMT5-C; A). Equal volumes of the
gradient samples were subjected to the MBP-specific methyltransferase
assay. Reaction products were visualized by SDS-PAGE and fluorography
(C), and 14C incorporation was quantified using
a PhosphorImager (B). Molecular mass markers (Combitek)
sedimented in a parallel sucrose gradient are indicated.
14C-Methylated MBP (MBP) and endogenous proteins
are indicated by arrowheads in C. Mu,
multimer.
View larger version (32K):
[in a new window]
Fig. 8.
Comparison of the specific enzyme activity of
purified FLAG-PRMT5 and FLAG-PRMT1. In vitro
methylation reactions were performed with 1 µg of GST-GAR and various
concentrations of purified FLAG-PRMT5 or FLAG-PRMT1 in the presence of
0.25 µCi of [14C]AdoMet. The reaction products were
analyzed by SDS-PAGE and fluorography and quantified using a
PhosphorImager.
View larger version (53K):
[in a new window]
Fig. 9.
PRMT5 is predominantly localized in the
cytoplasm. The gene for green fluorescent protein was fused in
frame to the N terminus of PRMT5. COS-1 (A) and Chang liver
(B) cells cultured on the coverslips were transfected with
the GFP or GFP-PRMT5 expression plasmid. The cells were photographed at
800-fold magnification under a confocal microscope. The fluorescent
regions are indicated by arrows.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Harvey R. Herschman for the pGST-PRMT1 and pGST-GAR constructs, Meungwon Kim for assistance with plasmid construction and protein purification, and J. Y. Kim and K. H. Kim for excellent technical help.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Ministry of Science and Technology, Korea. Amino acid analysis was supported by a program using the common equipment of the Korea Basic Science Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
82-42-860-4172; Fax: 82-42-860-4597; E-mail:
imdongsu@mail.kribb.re.kr.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M008660200
2 J. Rho, S. Choi, Y. R. Seong, J. Choi, and D.-S. Im, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PRMTs, protein-arginine methyltransferases; MPB, myelin basic protein; HCV, hepatitis C virus; GST, glutathione S-transferase; GFP, green fluorescent protein; AdoMet, S-adenosyl-L-methionine; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; HPLC, high pressure liquid chromatography.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gary, J. D., and Clarke, S. (1998) Prog. Nucleic Acids Res. Mol. Biol. 61, 65-131[Medline] [Order article via Infotrieve] |
2. |
Najbauer, J.,
Johnson, B. A.,
Young, A. L.,
and Aswad, D. W.
(1993)
J. Biol. Chem.
268,
10501-10509 |
3. | Liu, Q., and Dreyfuss, G. (1995) Mol. Cell. Biol. 15, 2800-2808[Abstract] |
4. | Kim, S., Merrill, B., Rajpurohit, R., Kumar, A., Stone, K., Papov, V., Schneiders, J., Szer, W., Wilson, S., Paik, W. K., and Williams, K. (1997) Biochemistry 36, 5185-5192[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Smith, J., J.,
Rucknagel, K. P.,
Schierhorn, A.,
Tang, J.,
Nemeth, A.,
Lindert, M.,
Herschmann, H., R.,
and Wahle, E.
(1999)
J. Biol. Chem.
274,
13229-13234 |
6. | Baldwin, G. S., and Carnegie, P. R. (1971) Science 171, 579-581[Medline] [Order article via Infotrieve] |
7. | Stoner, G. L. (1984) J. Neurochem. 43, 433-447[Medline] [Order article via Infotrieve] |
8. |
Brahms, H.,
Raymackers, J.,
Union, A.,
de Keyser, F.,
Meheus, L.,
and Luhrmann, R.
(2000)
J. Biol. Chem.
275,
17122-17129 |
9. |
Lin, W.-J.,
Gary, D. J.,
Yang, M. C.,
Clarke, S.,
and Herschmann, H. R.
(1996)
J. Bio. Chem.
271,
15034-15044 |
10. |
Tang, J.,
Gary, D. J.,
Clarke, S.,
and Herschmann, H. R.
(1998)
J. Biol. Chem.
273,
16935-16945 |
11. | Stallcup, M. R., Chen, D., Koh, S. S., Ma, H., Lee, Y.-H., Li, H., Schurter, B. T., and Aswad, D. W. (2000) Biochem. Soc. Trans. 28, 415-418[Medline] [Order article via Infotrieve] |
12. | Paik, W. K., and Kim, S. (1967) Biochem. Biophys. Res. Commun. 29, 14-20[Medline] [Order article via Infotrieve] |
13. |
Paik, W. K.,
and Kim, S.
(1968)
J. Biol. Chem.
243,
2108-2114 |
14. | Rouault, J. P., Falette, N., Guehenneux, F., Guillot, C., Rimokh, R, Wang, Q., Berthet, C., Moyret-Lalle, C., Savatier, P., Pain, B., Shaw, P., Berger, R., Samarut, J., Magaud, J. P., Ozturk, M., Samarut, C., and Puisieux, A. (1996) Nat. Genet. 14, 482-486[Medline] [Order article via Infotrieve] |
15. | Montagnoli, A., Guardavaccaro, D., Starace, G., and Tirone, F. (1996) Cell Growth Differ. 7, 1327-1336[Abstract] |
16. |
Tang, J.,
Frankel, A.,
Cook, R. J.,
Kim, S.,
Paik, W. K.,
Williams, K. R.,
Clarke, S.,
and Herschmann, H. R.
(2000)
J. Biol. Chem.
275,
7723-7730 |
17. |
Tang, J.,
Kao, P. N.,
and Herschmann, H. R.
(2000)
J. Biol. Chem.
275,
19866-19876 |
18. |
Abramovich, C.,
Yakobson, B.,
Chebath, J.,
and Revel, M.
(1997)
EMBO J.
16,
260-266 |
19. | Katsanis, N., Yaspo, M.-L., and Fisher, E. M. C. (1997) Mamm. Genome 8, 526-529[CrossRef][Medline] [Order article via Infotrieve] |
20. | Scott, H. S., Antonarakis, S. E., Lalioti, M. D., Rossier, C., Silver, P. A., and Henry, M. F. (1998) Genomics 48, 330-340[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Frankel, A.,
and Clarke, S.
(2000)
J. Biol. Chem.
275,
32974-32982 |
22. |
Chen, D.,
Ma, H.,
Hong, H.,
Koh, S. S.,
Huang, S.-M.,
Schurter, B. T.,
Aswad, D. W.,
and Stallcup, M. R.
(1999)
Science
284,
2174-2177 |
23. |
Pollack, B. P.,
Kotenko, S. V.,
He, W.,
Izotova, L. S.,
Barnoski, B. L.,
and Pestka, S.
(1999)
J. Biol. Chem.
274,
31531-31542 |
24. |
Gilbreth, M.,
Yang, P.,
Wang, D.,
Frost, J.,
Polverino, A.,
Cobb, M. H.,
and Marcus, S.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
13802-13807 |
25. |
Gilbreth, M.,
Yang, P.,
Bartholomeusz, G.,
Pimental, R. A.,
Kansra, S.,
Gadiraju, R.,
and Marcus, S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14781-14786 |
26. | Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., and Houghton, M. (1989) Science 244, 359-362[Medline] [Order article via Infotrieve] |
27. | Saito, I., Miyamura, T., Ohbayashi, A., Harada, H., Katayama, T., Kikuchi, S., Watanabe, Y., Koi, S., Onji, M., Ohta, Y., Choo, Q. L., Houghton, M., and Kuo, G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6547-6549[Abstract] |
28. | Bartenschlager, R., Ahlborn-Laake, L., Mous, J., and Jacobson, H. (1993) J. Virol. 67, 3835-3844[Abstract] |
29. | Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M., and Rice, C. M. (1993) J. Virol. 67, 2832-2843[Abstract] |
30. | Hijikata, M., Mizhushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., Tanaka, T., Kimura, K., and Shimotohno, K. (1993) J. Virol. 67, 4665-4675[Abstract] |
31. | Tomei, L., Failla, C., Santolini, E., De Franscesco, R., and La Monica, N. (1993) J. Virol. 67, 4017-4026[Abstract] |
32. | Kim, D. W., Gwack, Y., Han, J. H., and Choe, J. (1997) J. Virol. 71, 9400-9409[Abstract] |
33. |
Lin, C.,
and Kim, J. L.
(1999)
J. Virol.
73,
8798-8807 |
34. | Sakamuro, D., Furukawa, T., and Takegami, T. (1995) J. Virol. 69, 3893-3896[Abstract] |
35. | Fujita, T., Ishido, S., Muramatsu, S., Itoh, M, and Hotta, H. (1996) Biochem. Biophys. Res. Commun. 229, 825-831[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Ghosh, S. K.,
Paik, W. K.,
and Kim, S.
(1988)
J. Biol. Chem.
263,
19024-19033 |
37. | Rawal, N., Rajpurohit, R., Paik, W. K., and Kim, S. (1994) Biochem. J. 300, 483-489[Medline] [Order article via Infotrieve] |
38. | Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. (1993) Cell 75, 791-803[Medline] [Order article via Infotrieve] |
39. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
40. | Matsumoto, M., Hwang, S. B., Jeng, K.-S., Zhu, N., and Lai, M. M. C. (1996) Virology 218, 43-51[CrossRef][Medline] [Order article via Infotrieve] |
41. | Koonin, E. V. (1993) J. Gen. Virol. 74, 733-740[Abstract] |
42. | Kagan, R. M., and Clarke, S. (1994) Arch. Biochem. Biophys. 310, 417-427[CrossRef][Medline] [Order article via Infotrieve] |
43. | Schluckebier, G., O'Gara, M., Saenger, W., and Cheng, X. (1995) J. Mol. Biol. 247, 16-20[CrossRef][Medline] [Order article via Infotrieve] |
44. | Lischwe, M. A., Ochs, R. L., Reddy, R., Cook, R. G., Yeoman, L. C., and Busch, H. (1985) Biochemistry 24, 6025-6028[Medline] [Order article via Infotrieve] |
45. | Lapeyre, B., Caizergues-Ferrer, M., Bouche, G., and Amalric, F. (1985) Nucleic Acids Res. 13, 5805-5816[Abstract] |
46. | Heine, M. A., Rankin, M. L., and Dimario, P. J. (1993) Mol. Biol. Cell 4, 1189-1204[Abstract] |
47. |
Gary, J. D.,
Lin, W.-J.,
Yang, M. C.,
Herschmann, H. R.,
and Clarke, S.
(1996)
J. Biol. Chem.
271,
12585-12594 |
48. | Kim, S., Lim, I. K., Park, G.-H., and Paik, W. K. (1997) Int. J. Biochem. Cell Biol. 29, 743-751[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Krapivinsky, G.,
Pu, W.,
Wickman, K.,
Krapivinsky, L.,
and Clapham, D. E.
(1998)
J. Biol. Chem.
273,
10811-10814 |
50. | Krapivinsky, G. B., Ackerman, M. J., Gordon, E. A., Krapivinsky, L. D., and Clapham, D. E. (1994) Cell 76, 439-448[Medline] [Order article via Infotrieve] |
51. | Darnell, J. E., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415-1421[Medline] [Order article via Infotrieve] |
52. | Winston, L. A., and Hunter, T. (1996) Cell. Signal. 7, 739-745[CrossRef] |
53. | Ihle, J. N. (1995) Adv. Immunol. 60, 1-35[Medline] [Order article via Infotrieve] |
54. | Muramatsu, S., Ishido, S., Fujita, T., Itoh, M., and Hotta, H. (1997) J. Virol. 71, 4954-4961[Abstract] |