From the Institut für Biochemie,
Martin-Luther-Universität Halle-Wittenberg, 06120 Halle, Germany,
§ Institut für Biochemie,
Justus-Liebig-Universität Giessen, 35392 Giessen, Germany,
¶ Max-Planck-Forschungsstelle für Enzymologie der
Proteinfaltung, 06120 Halle, Germany,
Molecular Biology
Institute, Department of Biological Chemistry, UCLA, Los Angeles,
California 90095, and
Biochemisches
Institut, Justus-Liebig-Universität Giessen,
35392 Giessen, Germany
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ABSTRACT |
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Arginine methylation is a post-translational
modification found mostly in RNA-binding proteins. Poly(A)-binding
protein II from calf thymus was shown by mass spectrometry and
sequencing to contain
NG,NG-dimethylarginine
at 13 positions in its amino acid sequence. Two additional arginine
residues were partially methylated. Almost all of the modified residues
were found in Arg-Xaa-Arg clusters in the C terminus of the protein.
These motifs are distinct from Arg-Gly-Gly motifs that have been
previously described as sites and specificity determinants for
asymmetric arginine dimethylation. Poly(A)-binding protein II and
deletion mutants expressed in Escherichia coli were
in vitro substrates for two mammalian protein arginine methyltransferases, PRMT1 and PRMT3, with
S-adenosyl-L-methionine as the methyl group
donor. Both PRMT1 and PRMT3 specifically methylated arginines in the
C-terminal domain corresponding to the naturally modified sites.
Poly(A)-binding protein II
(PABP2)1 is a protein of 33 kDa that binds poly(A) with high affinity and specificity. PABP2 is thought to be involved in pre-mRNA polyadenylation. In
vitro, it stimulates poly(A) polymerase, conferring processivity
on the reaction, and is responsible for poly(A) tail-length control
(1-3). The protein is composed of an acidic N terminus, a
ribonucleoprotein (RNP)2-type
RNA binding domain in the center, and an arginine-rich C terminus (4).
Both the RNP domain and the C terminus contribute to RNA
binding.3
A well known arginine-rich RNA binding domain is the RGG domain. The
RGG motif is defined as a variable number of closely spaced Arg-Gly-Gly
(RGG) repeats interspersed with other, often aromatic amino acids (5,
6). RGG motifs are normally found in conjunction with RNP domains (7,
8) and have been shown to increase the affinity of a protein for RNA
(9-13). It is possible that RGG motifs also confer specific RNA
binding to a protein, since heterogeneous nuclear RNP, protein U has no
other discernible RNA binding motif and can discriminate between
different RNA sequences (5). Other functions of the RGG domain thus far
described include mediation of protein-protein interactions (14, 15)
and of nuclear localization (16, 17).
A characteristic feature of the RGG motif is the post-translational
modification of arginine residues to
NG,NG-dimethylarginine
(NG,NG-DMA) (18-23). This modification is
carried out by protein arginine methyltransferases (PRMTs) using
S-adenosyl-L-methionine as a methyl donor. Rat
PRMT1 (24) and the human homologue HRMT1L2 (25) methylate RGG
motif-containing proteins and homologous synthetic peptides in
vitro to give
NG,NG-DMA and/or
monomethylarginine (MMA) residues. A second mammalian methyltransferase
termed PRMT3 has been cloned. Although this enzyme methylates a
glutathione-S-transferase (GST) fusion protein containing a
glycine- and arginine-rich region from human fibrillarin, GST-GAR, no
natural protein substrates have yet been identified for PRMT3 in heated
hypomethylated rat cell extracts. PRMT3 is predominantly cytoplasmic,
whereas PRMT1 is largely confined to the nucleus (26). Sequence
comparison of mapped DMA residues showed the preferred amino acid
sequence Phe/Gly-Gly-Gly-Arg-Gly-Gly/Phe with the C-terminal-flanking
glycine being invariant (22). It is unclear which enzyme is responsible
for the modification of this sequence in mammalian cells in
vivo (see "Discussion"). In contrast, the yeast arginine
methyltransferase Rmt1p is known to be responsible for arginine
dimethylation in vivo (27, 28). One identified in
vivo substrate is the RGG domain containing RNA binding protein
Npl3p (28, 29).
The biological function of arginine methylation is unknown. A role in
signal transduction has been suggested (24, 30, 31). However, the
modification may be irreversible: the amino-alkyl bond is very stable,
and there is no evidence of a DMA demethylase. Other suggested
functions include modulation of intracellular trafficking of
macromolecules (32-34) and of protein binding to RNA (35). A yeast
rmt1 deletion strain is viable under standard growth
conditions (27, 28).
Upon amino acid analysis of PABP2 from calf thymus, only 50% of the
expected arginine residues were found (4). Here we show that arginine
is methylated at 15 locations in PABP2. Almost all modified arginines
are located in the C terminus of the protein in sequence motifs
distinct from previously described sites of arginine methylation. The
mammalian arginine methyltransferases PRMT1 and PRMT3 preferentially
methylate the C terminus of recombinant PABP2 in vitro.
Purification of Proteins--
PABP2 was purified from calf
thymus as described (3). PABP2 used for enzymatic digests was further
purified on a Nucleosil 500-5 C3-PPN HPLC column (150 × 2 mm) (Macherey-Nagel, Düren, Germany) equilibrated with
0.09% trifluoroacetic acid and eluted by a 30-60% solvent B (0.08%
trifluoroacetic acid in acetonitrile) gradient over 25 min with a flow
rate of 0.2 ml/min at 40 °C. The identity of the PABP2 peak was
confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
and the fraction was dried under a stream of nitrogen. Cysteine
residues were alkylated with vinylpyridine as described (36). The
alkylated protein was desalted by HPLC and dried as above. GST-PRMT
fusion proteins were purified as described (24, 26).
Preparation of Recombinant PABP2 and Deletion Mutants--
The
PABP2 coding sequence from a pT7-7-PABP2 construct (4) was cloned into
the NdeI and BamHI sites of the
pGM10(His)6 expression vector (37). From this construct a
fusion protein with the sequence Met-Ala-(His)6-PABP2 was
expressed. Deletion mutants were obtained through polymerase chain
reaction using pT7-7-PABP2 as a template and suitable oligonucleotide
primers. PABP2 Enzymatic Digestion of PABP2--
Sequencing grade proteases
were obtained from Roche Molecular Biochemicals. Lys-C digestion of
PABP2 was carried out in 50 µl of 25 mM Tris-HCl, pH 8.5, with a Lys-C to PABP2 ratio of 1:200 (w/w) overnight at 37 °C.
Digests were separated by HPLC as above with a 0-40% solvent B
gradient over 60 min and a flow rate of 0.2 ml/min at 40 °C.
Fragments labeled Lys-C1-8 in the order of elution (Table I) were
dried under a nitrogen stream and analyzed by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
Secondary chymotryptic digestion was carried out on Lys-C6 in 100 mM NH4HCO3, pH 8.5, with a
chymotrypsin to protein ratio of around 1:100 (w/w) for 4 h at
room temperature. Peptides were separated by HPLC and analyzed by
MALDI-TOF-MS. Chymotryptic fragments are labeled Chym followed by the
name of the digested substrate. Secondary tryptic digestion was carried out on Lys-C5b, Lys-C5c, and Lys-C8 in 100 mM
NH4HCO3, pH 8.5, with a trypsin to protein
ratio of 1:20 (w/w). Complete digests were analyzed directly by
MALDI-TOF-MS after 2 and 14 h of digestion. Trypsin fragments are
labeled Tryp followed by the name of the digested substrate.
Carboxypeptidase B type I diisopropyl fluorophosphate (Sigma) was
purchased as a frozen solution. 1 µl of sample was incubated with 0.4 µl of a 1:1000 dilution (50 mM
NH4HCO3 buffer) of the carboxypeptidase B stock
solution for 1 h at room temperature.
Mass Spectrometry--
Mass spectra were recorded on a REFLEX
MALDI-TOF mass spectrometer with SCOUT ion source and pulsed ion
extraction (Bruker-Franzen, Bremen, Germany). Data were analyzed with
the XMASS software supplied with the spectrometer. For analysis of
peptides, Amino Acid Sequencing--
Amino acid sequencing was carried out
with an Applied Biosystems 476A sequencer according to the
manufacturer's instructions. MMA,
NG-NG'-DMA (symmetric)
and NG-NG-DMA
(asymmetric) (Sigma) were directly applied to the sequencer filter, and
after one cycle the phenylthiohydantoin amino acid retention times were
obtained. These were used for comparison with retention times of
peptide-derived phenylthiohydantoin amino acids.
Location of Modified Arginine Residues in PABP2 from Calf
Thymus--
We set out to identify the nature of arginine
modifications and to map their locations in the entire PABP2 sequence
by proteolytic digestion of the protein and subsequent analysis of the
derived fragments by sequencing and MALDI-TOF-MS. Lys-C fragments that could be assigned to the PABP2 sequence were labeled Lys-C1-8 in the
order of elution from the HPLC column (Table
I). The fragments designated Lys-C5b and
Lys-C5c were shoulders eluting after the main peak Lys-C5a. The entire
PABP2 sequence could be accounted for except for three small peptides
(Leu-136
Fragments Lys-C1, Lys-C3, and Lys-C4 did not contain arginine in their
sequences. The fragment Lys-C2 assigned to Ala-124
Fifteen sites of arginine modification were found in the remaining
PABP2 fragments by mass spectral analysis and sequencing. The largest
discrepancy between observed and predicted mass was seen in the
C-terminal fragment Lys-C6, corresponding to Arg-248
Some heterogeneity in the extent and nature of arginine modification
was observed at two locations. The two peptides Lys-C5b and Lys-C5c,
eluted from the HPLC column as shoulders of Lys-C5a, were, based on
their masses, assigned to Glu-224
Fragment Lys-C8, corresponding to the N terminus of the protein, had an
observed mass of 11,836.7 Da. The predicted mass was 11930.7 Da. The N
terminus of the protein was blocked, and the mass discrepancy infers
removal of the initial methionine residue and N terminus acetylation.
However, the high mass of this peak made discernment of partial
modification with a single methyl residue unlikely, so a secondary
tryptic digest was carried out. Fragments obtained from this digest
(Tryp-Lys-C8) included minute amounts of two peptides corresponding to
the sequence acetyl
The measured mass of the entire HPLC-purified protein was 33,253 Da.
This is in excellent agreement with the predicted mass of 33,252.5 Da,
assuming removal of the initial methionine, acetylation, alkylation of
two cysteine residues, and the addition of 26 methyl groups.
E. coli-expressed PABP2 and Deletion Mutants Are Substrates for the
Arginine Methyltransferases PRMT1 and PRMT3--
The two mammalian
methyltransferases PRMT1 and PRMT3, expressed in E. coli as
GST fusion proteins (24, 26), were able to methylate E. coli-expressed PABP2 and various deletion mutants with
S-adenosyl-L-methionine as the methyl group
donor (Fig. 2). As expected, the best
substrates were those that contained the C terminus of the protein.
Both the N-terminal deletion mutant (PABP2
Lys-C digestion of full-length recombinant PABP2 methylated in
vitro by GST-PRMT1 followed by MALDI-TOF-MS analysis indicated a
shift of the C-terminal fragment to a higher mass (Fig.
3, b and d). No
additional peaks corresponding to methylated species were found in any
other peptide (compare with Fig. 3, a and c); all
observed masses were in close agreement with predicted values (data not
shown). The C-terminal peak was centered at around 7069 mass units,
corresponding to the addition of four methyl groups per fragment. The
mass distribution of the C-terminal fragment could be modeled with an
average of 0.22 methyl groups/arginine residue (data not shown). Taking
into account this level of methylation, the resolution of MALDI-TOF MS
and the number of arginines in the different peptides, and assuming
random methylation, methylated species of the peptides corresponding to
Lys-C2, Lys-C7, and Lys-C5 should have been detectable. These species
were not observed, indicating that methylation in vitro was
preferentially directed to the C terminus. Preferential methylation of
the C terminus upon in vitro methylation of PABP2 by
GST-PRMT3 was also observed (data not shown).
Previous studies on the substrate specificity of arginine
methylation, based on sequence comparison of the 20 thus far mapped sites of arginine dimethylation, described a preferred recognition motif of Phe/Gly-Gly-Gly-Arg-Gly-Gly/Phe with the C-terminal-flanking glycine considered obligatory (22, 23, 39). In PABP2, only 3 of the 13 identified sites of complete arginine dimethylation have a
C-terminal-flanking glycine, and of these three, none have an
N-terminal-flanking glycine. Arg-17 has N- and C-terminal-flanking glycine residues, but only a small subpopulation of Arg-17 is mono- or
dimethylated. Twelve of the modified residues occur within a distinct
RXR motif, where both arginines in the motif are completely asymmetrically dimethylated, and X is, in most cases, a
small amino acid (Gly, Ala, Ser, or Pro) or, in one case, tyrosine.
PABP2 contains three RXR motifs (Arg-23 A data base search for other proteins containing an
RXRXRXR sequence with Pro, Tyr, Ala,
or Gly in the X position identified a number of nuclear
proteins including high molecular weight basic fibroblast growth factor
and ribosomal protein S2. High molecular weight basic fibroblast growth
factor has been shown to contain DMA within its RG motifs (32, 40, 41),
although the exact nature of the arginine modification has yet to be
established. Ribosomal protein S2 has RGGF and RGR motifs and is likely
to contain DMA (42). ICP27, an RNA-binding protein from herpesvirus, has RGR repeats in its sequence and is methylated in vivo,
although the sites of methylation have not been identified (17). If one includes serine in the X position in the data base search, a
great many splicing factors with their characteristic SR repeats are found. In the case of PABP2 amino acids, Ser-276 The RGG domain of nucleolin, which is built up of RGGF repeats and
contains NG,NG-DMA, has
been suggested to form repeated type I The finding that two different sequences, RXR clusters and
(F/G)GGRGG(G/F) (see above), are subject to asymmetric arginine dimethylation might suggest that each of the two identified mammalian arginine methyltransferases serves one of these two substrates. However, the situation is far from clear. Most studies of arginine methylation in vitro have been carried out with enzyme
preparations from mammalian tissue (23, 39, 46, 47). Their molecular compositions were ill-defined, and their relationship to PRMT1 and -3 is unknown. The two cloned methyltransferases, PRMT1 and PRMT3, may be
just the catalytic subunits of larger complexes (23). Moreover,
in vitro arginine methylation reactions are generally
plagued by poor efficiency. Thus, conclusions concerning substrate
specificity may be premature. The activity of both PRMT1 and PRMT3
in vitro was directed toward the C terminus of PABP2, with
PRMT3 apparently being more efficient than expected from previous
experiments with a standard RGG substrate. This suggests that both
enzymes may have specificity for RXR clusters. There is a
possibility that this apparent specificity is directed by the structure
of the substrate protein rather than a preference of the enzymes for
particular amino acid sequences. However, in a comparison of several
synthetic peptides, a peptide with clustered RGR sequences was by far
the best in vitro substrate for arginine methyltransferase
preparations from three different tissues (38).
Biochemical assays of PABP2 have so far not revealed any function for
arginine methylation. Recombinant and authentic proteins are
indistinguishable with respect to the stimulation of polyadenylation and length control (4). Under standard reaction conditions, we have
also not detected any difference in the RNA binding properties of the
two proteins.3
In addition to some similarity in the general amino acid composition,
the C-terminal RXR domain of PABP2 shares two further features with the RGG domain in that it contains the characteristic modified amino acid asymmetric dimethylarginine, and it binds RNA in a
nonsequence specific manner.3 Structural studies are needed
to decide whether RXR and RGG sequences are merely two
variations on the same theme or two different motifs fulfilling similar roles.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C contained PABP2 amino acids 1 through 257, PABP2
N amino acids 161 through 306, RNP domain amino acids 161 through
257, and the C-terminus amino acids 258 through 306. Polymerase chain reaction products were cut with NdeI and BamHI
and cloned into pGM10(His)6 so that each protein was
expressed with the N-terminal sequence Met-Ala-His6.
Constructs were checked by sequencing. Proteins were expressed in
Escherichia coli BL21-pLys-S in Superbroth medium. 2-liter
cultures with an A600 of around 2 were induced with 0.4 mM isopropyl
-D-thiogalactopyranoside and grown for 4 h at
37 °C. Cells were pelleted, stored overnight at
80 °C, and
suspended in 50 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM KCl, 10% (v/v) glycerol). This and all subsequent
buffers contained 0.5 mM phenylmethylsulfonyl fluoride, 0.4 µg/ml leupeptin, and 0.7 µg/ml pepstatin. Lysates were prepared by
sonication for 5 min, cleared by centrifugation, and incubated for
2 h with 1 ml nickel nitrilotriacetic acid-agarose (Qiagen)
pre-equilibrated with lysis buffer. The slurry was loaded into a column
and washed with 10 ml of lysis buffer and then with 10 ml of lysis
buffer containing 10 mM imidazole. His6-tagged
proteins were eluted with 5 × 1-ml fractions of lysis buffer
containing 250 mM imidazole. PABP2-containing fractions
were dialyzed against 1 liter of 25 mM HEPES, pH 7.9, 10%
glycerol, 10 mM KCl, 0.5 mM dithiothreitol. Proteins were loaded onto a Mono S column (Amersham Pharmacia Biotech)
in dialysis buffer and eluted with a 0-100% gradient of the same
buffer containing 1 M KCl. PABP2-containing fractions were
dialyzed against 25 mM Tris-HCl, pH 8.0, 10% glycerol, 10 mM KCl, 0.5 mM dithiothreitol, and frozen in
liquid nitrogen. The PABP2 C terminus was purified by Ni2+
chelate chromatography under denaturing conditions according to the
manufacturer's instructions.
-cyano-4-hydroxycinnamic acid and nitrocellulose were
dissolved in acetone to a concentration of 20 and 5 g/liter,
respectively. 1 µl of this solution was deposited on a stainless
steel sample stage. 1 µl of peptide solution was spotted onto this
matrix surface. Protein analysis was carried out using sinapinic acid
as matrix. A layer of a saturated solution of sinapinic acid (39:60:1
acetone, methanol, 0.1% trifluoroacetic acid (v/v/v)) was applied to
the sample stage. A mixture of 1 µl of the protein and 1 µl of a
second saturated sinapinic acid solution (30:20:50
acetonitrile:methanol:water (v/v/v)) was then applied on the matrix
layer. All spectra were calibrated using external standards.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Lys-137, Phe-208
Lys-213 and Val-244
Lys-247), none of
which contained arginine. The cDNA-derived PABP2 amino acid
sequence with the sites of modification is shown in Fig.
1. All residue numbering is according to
this sequence. For an overview of the fragments, their masses, and
assignments, see Table I. Sequences obtained from individual fragments
are listed in Table II.
Origin, assignment, and mass of PABP2 proteolytic digestion fragments
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Fig. 1.
cDNA sequence of PABP2 from Bos
taurus. Arginine residues found to be fully
asymmetrically dimethylated by sequencing appear in black-shaded
boxes. Partially methylated arginine residues appear in
gray-shaded boxes. The underlined residues
delineate the RNP domain.
Obtained sequences of PABP2 proteolytic digestion fragments
Lys-135 contained
two arginine residues, Arg-125 and Arg-127. Lys-C5a was assigned to
Glu-224
Lys-243. This assignment was confirmed by sequencing. The
fragment contained three arginine residues, Arg-227, Arg-238, and
Arg-240. The observed masses of all the above-mentioned fragments were
in very close agreement with the masses predicted from the cDNA
sequence, indicating no modification of amino acid residues. Lys-C5b
and Lys-C5c are discussed below. Fragment Lys-C7, corresponding to
Met-167-Lys-207, contained two arginine residues, Arg-172 and Arg-200.
This fragment also contained two cysteine residues, Cys-195 and
Cys-205. The observed mass of this fragment corresponded to the
predicted mass plus two pyridylethyl cysteine modifications, indicating
that the two arginine residues present are unmodified.
Tyr-306. The
difference, 364.8 mass units, corresponds to the addition of 26 methyl
groups, assuming the only modification present is methylation. The
predicted sequence of the fragment contains 15 arginine residues. Upon
sequencing of the first 35 amino acids in Lys-C6, the initial two
arginine residues, Arg-248 and Arg-251, were found to be unmodified,
whereas arginines 259, 263, 265, 267, 269, 277, and 279 were found to
be dimethylated. Phenylthiohydantoin derivatives of the modified
arginines had the same retention time in the sequencer as an
NG-NG-DMA standard.
Sequences obtained from two secondary chymotryptic fragments, one
generated by incomplete digestion, confirmed the dimethylation of
arginines 287, 289, 291, 294, 296, and 298 (Table II).
Lys-243 with the addition of 1 and 2 methyl groups, respectively. The predicted sequence of the peptides
(ESVRTSLALDESLFRGRQIK) contained three arginine residues, any one of
which could be a site of modification. Sequencing confirmed the
identity of the peptides and identified their fourth amino acid as
unmodified arginine. Data quality was not sufficient for unequivocal
identification of the remaining arginines. To localize the site of
modification, a secondary tryptic digest was carried out on peptides
Lys-C5b and Lys-C5c. Mass spectral analysis of the entire digest gave
fragments with masses corresponding to the predicted sequence
TSLALDESLFRGR with the addition of one and two methyl groups for
Tryp-Lys-C5b and Tryp-Lys-C5c, respectively. Trypsin will not cleave
after a methylated arginine residue (22), and therefore it is assumed
that the site of modification in both Lys-C5b and Lys-C5c is Arg-238.
In the Lys-C5b tryptic digest, a significant peak with a mass of 1251 Da, corresponding to Thr-228
Arg-238 without modification, was also
observed, indicating that the separation of the two peptides Lys-C5a
and Lys-C5b was not complete. The relative amounts of the unmodified,
monomethylated, and dimethylated peptides, i.e. Lys-C5a,
Lys-C5b, and Lys-C5c, were approximately 2:1:1 based on HPLC peak heights.
Ala-2
Arg-23 with the addition of 1 or 2 methyl
groups. The full predicted sequence of these fragments was
acetyl-AAAAAAAAAAGAAGGRGSGPGR. As in the case of Lys-C5b and Lys-C5c,
the presence of methylated arginine groups in a tryptic peptide ending
with arginine infers that the modification occurs on the interior
arginine, in this case Arg-17. Further evidence was obtained by
treatment of the entire tryptic digest with carboxypeptidase B. Mass
spectral analysis after this treatment gave fragments with masses of
the original fragments less the mass of one unmodified arginine
residue. In contrast to Arg-238, which was modified to about 50%, a
very small proportion (<5%) of Arg-17 was found to be methylated. We
cannot be certain as to the exact identity of the modified residues at Arg-17 and Arg-238, as these fragments could not be sequenced. However,
as NG-NG'-DMA
(symmetric) has thus far only been found in myelin basic protein (23,
38), it is highly unlikely that the modified arginine residues at
Arg-17 and Arg-238 are the symmetric form of DMA. Arg-24 and Arg-25
could not be assigned to any tryptic fragment from the Tryp-Lys-C8
digest. This suggests that they are unmodified arginine residues that
served as tryptic cleavage sites. It is also possible that Arg-24 is
modified to a very small extent, and Arg-23 and Arg-25 serve as tryptic
cleavage sites. The resulting dipeptide Arg-24
Arg-25 would not be
detected by MALDI-TOF-MS.
N) and the isolated
C-terminal domain (Fig. 2, b and c, lanes
4 and 5) appeared to be better substrates for both
PRMT1 and PRMT3 than the complete protein (lane 1). In the
experiment shown in Fig. 2, use of PRMT3 at an approximately 2.5-fold
higher molar concentration than PRMT1 led to a similar extent of
methylation. In previous experiments (26), PRMT3 had approximately 1%
of the activity of PRMT1 when GST-GAR was used as a methyl-accepting substrate. Thus, the activity of the GST-PRMT3 protein toward PABP2 was
higher than expected. Extra methylated bands in Fig. 2, b
and c, are presumably contaminants from the substrate or PRMT fusion protein purification. Their identities are unknown.
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Fig. 2.
PABP2 and deletion mutants are methylated
in vitro by PRMT1 and PRMT3. a,
schematic of PABP2 deletion mutant constructs and the efficiency of
their in vitro methylation by GST-PRMT1 and GST-PRMT3.
b, in vitro methylation of PABP2 and deletion
mutants (arrows) by GST-PRMT1. Methylation reactions
contained 0.65 µg (20 pmol) of recombinant PABP2 (lane 1),
1 µg (33 pmol) of PABP2 C (lane 2), 1 µg (100 pmol)
of RNP domain (lane 3), 0.65 µg (100 pmol) of C terminus
(lane 4), 0.65 µg (40 pmol) of PABP2
N (lane
5), 0.75 µM (3.3 µCi)
S-adenosyl-L-[methyl-3H]methionine,
and 0.5 µg of PRMT1 in a final volume of 55 µl with a buffer of 25 mM Tris-HCl, 1 mM sodium EDTA,1 mM
sodium EGTA at pH 7.5. c, identical reactions were carried
out with 3 µg of GST-PRMT3. Reactions, SDS-polyacrylamide gel
electrophoresis, and fluorography were carried out as described
previously (24, 26).
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Fig. 3.
In vitro methylation of PABP2
expressed in E. coli is directed to the C
terminus. MALDI-TOF-MS spectra of peptides corresponding to Lys-C5
(a and c) and Lys-C6 (b and
d). a and b control reactions without
methyltransferase; c and d , after incubation
with GST-PRMT1. The methylation reaction contained 2 µg of PABP2, 60 µM S-adenosyl-L-methionine, 0.5 µg of GST-PRMT1 in a buffer with 25 mM Tris-HCl, 1 mM EDTA, pH 8.0, in a total volume of 100 µl. The
reaction was incubated at 37 °C for 60 min. PABP2 purification by
HPLC, Lys-C digestion, and MALDI-TOF-MS were carried out as described
under "Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Arg-25,
Arg-76
Arg-78, and Arg-125
Arg-127) with unmethylated arginines. The
sequence RXR is thus not a sufficient determinant of
arginine dimethylation. As amino acids 76-78 are RPR, the identity of
X cannot be a sufficient determinant either. It remains to
be determined whether the additional criteria are the sequence context,
the accessibility of the arginines for the methyltransferase, or the
clustering of RXR motifs. The N-terminal deletion mutant was
a better substrate for both PRMT1 and PRMT3 than the entire protein. It
is possible that the acidic N terminus makes intra- or intermolecular
contacts with the basic C terminus, the disruption of which improves
accessibility for the methyltransferase. Note, however, that the
increased molar concentration of the smaller substrates may also have
promoted increased levels of methylation.
Arg-279 form an SRSR
motif in which both arginines are dimethylated. This raises the
possibility that splicing factors could serve as methyl-accepting substrates in vivo. In fact, the yeast protein Npl3p has
been shown to be subject to both arginine methylation and serine
phosphorylation (29). Although these modifications have not yet been
mapped, the protein contains a number of SRGG motifs, which might serve as targets for both. As yet, arginine dimethylation has not been detected in the well studied mammalian SR proteins.
-turns (9). Gly-239 of PABP2
corresponds to a conserved glycine residue that is found in most RNP
domains (6) and forms part of a type I
turn (6, 43-45). This amino
acid lies in the center of an RXR motif. Although Arg-238 is
only partially methylated, and Arg-240 is unmethylated, this
observation suggests that RXR motifs in the C terminus of
PABP2 might also form type I
-turns.
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ACKNOWLEDGEMENTS |
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We are grateful to Peter Bayer, Uwe Kühn, and Sylke Meyer for advice, discussions, and reading the manuscript. We thank Thomas Pfeifer, who carried out the carboxypeptidase B digest.
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FOOTNOTES |
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* This work was supported by the Training and Mobility of Researchers program of the European Union, the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie (to E. W.), and National Institutes of Health Grants GM24797 and AI34567 (to H. H. and J. T.).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.
** A postdoctoral trainee supported by USPHS Institutional National Research Service Award T32 CA09056.
§§ To whom correspondence should be addressed: Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany. Tel.: 49-345-55-24920; Fax: 49-345-55-27014; E-mail: ewahle{at}biochemtech.uni-halle.de.
1 PABP2 was formerly termed PABII. The name PABP2 has now been adopted from the human genome project nomenclature (48).
3 A. Nemeth, U. Kühn, and E. Wahle, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: RNP, ribonucleoprotein; DMA, dimethylarginine; GST, glutathione S-transferase; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MMA, monomethylarginine; PABP2, poly(A)-binding protein II; PRMT, protein arginine methyltransferase; HPLC, high performance liquid chromatography.
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