(Received for publication, January 11, 1996; and in revised form, March 4, 1996)
From the
We have identified the major enzymatic activity responsible for
the S-adenosyl-L-methionine-dependent methylation of
arginine residues (EC 2.1.1.23) in proteins of the yeast Saccharomyces cerevisiae. The RMT1 (protein-arginine
methyltransferase), formerly ODP1, gene product encodes a
348-residue polypeptide of 39.8 kDa that catalyzes both the N-mono- and N
,N
-asymmetric dimethylation
of arginine residues in a variety of endogenous yeast polypeptides. A
yeast strain in which the chromosomal RMT1 gene was disrupted
is viable, but the level of N
,N
-[
H]dimethylarginine
residues detected in intact cells incubated with S-adenosyl-L-[methyl-
H]methionine
is reduced to less than 15% of the levels found in the parent strain,
while the N
-[
H]monomethylarginine
content is reduced to less than 30%. We show that soluble extract from
parent cells, but not from mutant rmt1 cells, catalyzes the in vitro methylation of endogenous polypeptides of 55, 41, 38,
34, and 30 kDa. The hypomethylated form of these five polypeptides, as
well as that of several others, can be mono- and asymmetrically
dimethylated by incubating the mutant rmt1 extract with a
purified, bacterially produced, glutathione S-transferase-RMT1
fusion protein and S-adenosyl-L-[methyl-
H]methionine.
This glutathione S-transferase-RMT1 fusion protein is also
able to methylate a number of mammalian polypeptides including
histones, recombinant heterogeneous ribonucleoprotein A1, cytochrome c, and myoglobin, but cannot methylate myelin basic protein.
RMT1 appears to be a yeast homolog of a recently characterized
mammalian protein-arginine methyltransferase whose activity may be
modulated by mitotic stimulation of cells.
Evidence for the posttranslational methylation of arginine
residues in proteins was first provided by the presence of radioactive
species chromatographing at positions near that of arginine in acid
hydrolysates of isolated calf thymus nuclei incubated with S-adenosyl-L-[methyl-C]methionine
(Paik and Kim, 1967). The methylated species were later determined to
be arginine derivatives that had been mono- and dimethylated on their
guanidino group (Paik and Kim, 1968; Nakajima et al., 1971). S-Adenosyl-L-methionine-dependent methyltransferase
activities that catalyze these reactions have now been characterized in
a number of eukaryotic tissues and organisms.
When the partially
purified protein-arginine methyltransferase activity from calf brain
was incubated with histones as a methyl-accepting substrate, the
products of the reaction, after acid hydrolysis, were determined to be N-monomethylarginine, N
,N
-dimethylarginine
(asymmetric), and N
,N`
-dimethylarginine
(symmetric) (Lee et al., 1977) (see Fig. 1). However,
the substrate specificity of the enzyme at each stage of the
purification suggested that two distinct methyltransferases are
responsible for the creation of the three types of methylated species
(Miyake and Kakimoto, 1973; Lee et al., 1977). In support of
this conclusion, two distinct protein methylases have been partially
purified from calf brain. One enzyme activity specifically mono- and
symmetrically dimethylates myelin basic protein (Ghosh et al.,
1988) (Fig. 1) on arginine residue 107 (Baldwin and Carnegie,
1971; Brostoff and Eylar, 1971). The second activity was initially
described as a histone-specific methyltransferase (Ghosh et
al., 1988), although it is now known to be much more efficient for
substrates such as the heterogeneous ribonucleoprotein A1 (hnRNP A1) (
)and catalyzes the mono- and asymmetric dimethylation of
arginine residues (Fig. 1). The site of arginine methylation in
hnRNP A1 is within a domain that has been designated GAR for glycine
and arginine-rich and contains multiple repeats of a consensus arginine
methylation site RGG (Rajpurohit et al., 1994a). Several other
potential substrates for this enzyme also contain a similar GAR domain
(Najbauer et al., 1993). Members of this family of
methyl-accepting substrates include nucleolin and fibrillarin proteins
that contain 10-12 residues of asymmetric dimethylarginine
(Lischwe et al., 1985a; Lischwe et al., 1985b). These
proteins, like hnRNP A1, are involved in the processing of pre-RNAs.
Figure 1: Structures of N-methylated arginine residues found in proteins. Protein arginine methyltransferases catalyze the transfer of methyl groups from AdoMet to the terminal guanidino nitrogen atoms of arginine residues.
A number of purifications of a histone/hnRNP A1-specific mammalian arginine methyltransferase activity have been reported (Ghosh et al., 1988; Rawal et al., 1994; Liu and Dreyfuss, 1995). However, the polypeptide composition of this enzyme is still not established. Similarly, no genes encoding for a protein-arginine methyltransferase have been identified to date. Two purifications of the enzyme activity resulted in preparations that demonstrated multiple polypeptide species present after SDS-gel electrophoresis. Ghosh et al.(1988) identified two polypeptides (110 and 75 kDa) associated with a histone-specific arginine methyltransferase from calf brain. The purest fraction obtained from HeLa cells by Liu and Dreyfuss (1995) still had eight polypeptide bands present, with two prominent species of 100 and 45 kDa. A single 110-kDa polypeptide by SDS-gel electrophoresis was identified by Rawal et al.(1994) after a four-step purification from rat liver. However, only a small amount of material was analyzed by electrophoresis so that additional polypeptides of lower molecular mass may have escaped detection.
We have recently described the cDNA cloning and analysis of a mammalian gene product designated PRMT1 for protein- arginine methyltransferase (Lin et al., 1996). The 40.5-kDa protein encoded by this rat cDNA was found to interact, in a yeast two-hybrid screen (Fields and Song, 1989), with the murine primary response/immediate early gene product TIS21 (Fletcher et al., 1991; Varnum et al., 1994) and its family member the mouse antiproliferative gene product BTG1 (Rouault et al., 1992). A search of the sequence data base revealed that the mammalian PRMT1 gene product was similar in sequence to the yeast Saccharomyces cerevisiae ODP1 gene product. ODP1 has been identified as a partial open reading frame downstream of the PDX3 gene (Loubbardi et al., 1995), and its complete sequence was determined with the yeast chromosome II genomic sequencing effort (Feldmann et al., 1994). No function for ODP1 has been suggested. Using the combined biochemical and genetic approaches that can be taken in yeast, we were interested in exploring whether this gene might encode a protein-arginine methyltransferase with a function related to the mammalian enzyme. However, recent studies attempting to find an hnRNP A1 arginine methylating activity in yeast have been unsuccessful (Liu and Dreyfuss, 1995). Nevertheless, a search of the GenBank data base with the consensus arginine methylation sequence FGGRGGF has revealed numerous potential substrates for an enzyme of similar activity in yeast (Najbauer et al., 1993), including the nucleolin homolog NSR1, the fibrillarin homolog NOP1, as well as the SSB1, GAR1, and NPL3 proteins.
In this paper we provide evidence that the ODP1 gene product is, in fact, a protein-arginine methyltransferase that we now designate RMT1 (protein arginine methyltransferase). This enzyme can catalyze both the mono- and asymmetric dimethylation of the guanidino nitrogens of arginine residues present in a number of yeast polypeptides. We have constructed a yeast strain in which the RMT1 gene has been disrupted. The mutant cells are viable, but analysis of in vivo and in vitro methylated proteins from this strain demonstrated a dramatic decrease in the levels of mono- and asymmetrically dimethylated arginine residues present. The RMT1 gene product is therefore required for the majority of the mono- and asymmetric dimethylation of arginine residues in yeast. We also created and purified an N-terminal fusion of RMT1 with glutathione S-transferase. Incubation of the fusion protein with the methyl donor S-adenosyl-L-methionine results in the mono- and asymmetric dimethylation of the hypomethylated substrate proteins from the rmt1 soluble extract. These results demonstrate that the RMT1 gene product is a protein-arginine methyltransferase that plays a major role in modifying proteins containing a GAR domain, many of which interact with RNA. The methylation of arginine residues within these proteins may modulate this interaction, and may regulate other activities as well.
Figure 2: A schematic diagram of the wild-type RMT1, formerly ODP1, gene in S. cerevisiae, and of the constructed chromosomal insertion mutant. The lower portion represents a PCR product containing the complete coding region of RMT1. The initiator codon ATG is within the NdeI site and the HindIII site is 1 bp downstream of the termination codon TAA. Relevant restriction enzyme sites and the locations of conserved methyltransferase regions I, II, III, and post III (Kagan and Clarke, 1994; Kagan and Clarke, 1995) are included in the figure. The NdeI and HindIII sites were engineered into the 1.1-kbp fragment using primers RMT1-N1 and RMT1-C1, respectively, as described under ``Experimental Procedures.'' Arrowheads indicate the location of the 25-bp single-stranded DNA primers that were used for PCR analysis. The upper portion of the figure displays the insertional cassette from the plasmid YDp-LEU2 (Berben et al., 1991); the LEU2 coding region is represented by the filled arrow. The insertion of the LEU2 cassette into the unique BstXI site of RMT1 is described under ``Experimental Procedures.''
Plasmid pJG-RMT1 was digested at the unique BstXI site in the middle of the coding region (Fig. 2), and the 3` overhang was removed by subsequent treatment with T4 DNA polymerase. A LEU2 disruption cassette was obtained from plasmid YDp-LEU2 (Berben et al., 1991) by digestion with BamHI, and the resulting 5` overhang of the cassette was filled in by treatment with T4 DNA polymerase. The blunt-ended LEU2 cassette was then purified from a 1% agarose gel as described above and ligated into the BstXI-linearized pJG-RMT1 to create the disruption vector pJG-RMT1::LEU2. The orientation of the insertion was confirmed to be in the direction indicated in Fig. 2by restriction digests with NdeI/HindIII, NdeI/EcoRV, and SspI.
Replacement of the wild-type RMT1 chromosomal locus with the disruption construct was accomplished
in the strain CH9100-2 (MATa, prc1-407, prb1-1122, pep4-3, leu2, trp1, ura3-52, ycl57w::URA3)
(Hrycyna and Clarke, 1993) using the one-step technique described by
Rothstein (1983). Briefly, the disruption plasmid pJG-RMT1::LEU2 (10
µg) was digested with NdeI and HindIII, and the
entire mixture was used to transform CH9100-2 cells by the lithium
acetate method (Rose et al., 1990). The transformed cells were
then selected by plating onto leucine-deficient SCD plates (Rose et
al., 1990). Positives were rescreened on selective plates twice.
Genomic DNA was isolated from cells remaining after the three screens.
The replacement of the wild-type RMT1 locus by the LEU2 disrupted version was confirmed by PCR analysis using primers
RMT1-N2 (5`-TTCGTACCTTATCTTACAGAAACGC) and RMT1-C2
(5`-CAGTGAGTGTATGGAGCATGAGGAC) (Fig. 2). The wild-type locus
produces a PCR product of 700 bp, but the disrupted locus gives a
2.4-kbp product. All putative positives tested from the auxotrophic
screen produced only the 2.4-kbp product upon PCR analysis. The new rmt1 strain, in which the genomic copy of RMT1 has
been disrupted by a LEU2 cassette transcribed in the same
direction, is designated JDG9100-2 (MATa, prc1-407, prb1-1122, pep4-3, leu2, trp1, ura3-52, ycl57w
::URA3, rmt1::LEU2).
Figure 3: An alignment of the conserved methyltransferase regions from the S. cerevisiae RMT1, the rat protein-arginine methyltransferase PRMT1, and the E. coli L11 methyltransferase. The yeast RMT1 (PIR S45890) shares an overall 45% identity with its mammalian counterpart PRMT1 (Lin et al., 1996). The E. coli L11 protein methyltransferase (GenBank U18997, cf. GenBank Z26847) is the highest scoring protein found using a BLAST search with the RMT1 sequence and shares an 11% identity with RMT1. However, when methyltransferase regions I, post I, II, and III (Kagan and Clarke, 1994) for the three proteins are aligned, the sequences are highly conserved as is the inter-region spacing (not shown).
We then examined the endogenous protein methylation
patterns in the crude soluble fraction from both strains by incubating
these extracts with [H]AdoMet and then separating
the polypeptides by SDS gel electrophoresis (Fig. 4A).
At least five polypeptides (55, 41, 38, 34, and 30 kDa) that are
methylated in the lane containing parent RMT1 soluble extract
show little or no methylation in the mutant rmt1 soluble
extract lane. Therefore ODP1/RMT1 is required for a protein
methyltransferase activity in yeast.
Figure 4:
Loss of in vitro protein
methylation in soluble extract from the yeast rmt1 mutant strain. In panel A, cytosolic fractions containing parent extract (269
µg of protein) or mutant extract (338 µg of protein) (see
``Experimental Procedures'') were incubated in 0.8 µM [H]AdoMet (2.2 µCi) and a buffer of 25
mM Tris-HCl, 1 mM sodium EDTA, and 1 mM sodium EGTA at pH 7.5 in a final volume of 34 µl. After 30 min
at 30 °C, the reactions were stopped by adding an equal volume of 2
SDS-gel electrophoresis sample buffer (Hrycyna et al.,
1994). The samples were then separated by SDS-gel electrophoresis (10%
acrylamide) and the gel fluorographed as described under
``Experimental Procedures.'' This panel shows the result of a
2-month exposure at -80 °C. The arrows indicate the
position of molecular mass standards (Bio-Rad) for each gel (rabbit
phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; hen
egg ovalbumin, 42.7 kDa; bovine carbonic anhydrase, 31.0 kDa; soybean
trypsin inhibitor, 21.5 kDa; and hen egg lysozyme, 14.4 kDa). In panels B and C, endogenously methylated polypeptides
from either the parent (CH9100-2) or rmt1 mutant (JDG9100-2)
strain were analyzed by gel-slice methods. Reactions, prepared as
described above, contained either CH9100-2 cytosol (336 µg of
protein) or JDG9100-2 cytosol (423 µg of protein), 0.57 µM [
H]AdoMet (2.2 µCi), and buffer in a
final volume of 49 µl. After incubation and SDS-gel electrophoresis
as described above, gel slices were analyzed as described below. After
gel staining and destaining, each lane in the dried resolving gel was
cut into 35 separate 3-mm slices. In panel B, total
radioactivity was determined after each gel slice (with the filter
paper backing removed with forceps by wetting the paper with water) was
incubated with 1 ml of 30% (v/v) H
O
in a capped
scintillation vial at 70 °C for 24 h. After the gel dissolved, 5 ml
of scintillation fluid (Safety-Solve, Research Products International)
was added to the vial and the sample was counted to determine the
radioactivity present as methyl groups. In panel C, identical
slices from duplicate lanes were incubated with 100 µl of 2 N NaOH for 24 h at 70 °C. Using the vapor phase diffusion assay
described by Xie and Clarke(1993), the quantity of base-labile,
volatile methyl groups was determined. In both panels B and C, data from the parent strain is displayed with the open
circles, and data from the rmt1 mutant strain with closed circles. The arrows indicate the positions of
molecular mass standards described in panel
A.
To characterize the stability
of the [H]methylated residues, we compared the
total radioactivity present with the base-dependent volatile
radioactivity in parallel gel slices. The pattern of total radioactive
methyl group incorporation present in each slice (Fig. 4B) closely matches the data obtained from the
fluorograph, as expected (Fig. 4A). The major
methylated polypeptides at 55, 41, and 36 kDa from the endogenously
methylated parent soluble extract are again absent when extract
obtained from mutant rmt1 cells was used (Fig. 4B). The peak of radioactivity at 97 kDa and the
broad signal from 20-29 kDa are found in both the parent and the
mutant extracts and presumably represent methylation reactions not
dependent upon the ODP1/RMT1 gene product. Gel slices from
lanes parallel to those shown in Fig. 4B were then
analyzed after base treatment using a vapor phase diffusion assay (Fig. 4C) that detects [
H]methyl
groups in either ester linkages or linkages to the guanidino groups of
arginine residues (Paik and Kim, 1980; Najbauer et al., 1991;
Hrycyna et al., 1994). The results obtained from the analysis
of the parent cell extract indicate that the major 55-, 41-, and 36-kDa
substrates for the RMT1-dependent methyltransferase also represent the
majority of the base-labile, methylatable species present. To
distinguish volatile [
H]methylamine (derived from
[
H]methylarginine residues) from
[
H]methanol (derived from
[
H]methyl ester residues), we neutralized the
base-treated gel slices with HCl prior to the determination of
volatility. We found that the base-labile radioactivity derived from
the 55-, 41-, and 36-kDa polypeptides in the parent extract are not
observed under these conditions (data not shown), suggesting that the
product of the base treatment was, in fact,
[
H]methylamine as the methylammonium cation would
not be expected to be volatile.
The results shown in Fig. 4suggest that the yeast ODP1/RMT1 gene product is required for a large fraction of the total protein methylation as well as the predominant fraction of potential protein-arginine methylation reactions in vitro, since most of the base-labile methyl linkages observed are dependent upon the presence of RMT1. The remaining base-labile, volatile radioactivity seen at 42 kDa in the mutant extract in Fig. 4C may represent the C-terminal leucine methyl ester in the catalytic subunit of protein phosphatase 2A (Xie and Clarke, 1993, 1994). Similarly, the radioactivity peak in the 22-kDa region may represent the C-terminal isoprenylcysteine methyl esters in small G-proteins or the formation of methyl esters on tRNA molecules (Hrycyna et al., 1994).
Figure 5:
The in vivo mono- and asymmetric dimethylation of arginine
residues in yeast is dependent upon the RMT1 gene product. Panels A and C show the fraction of N,N
-dimethylated (asymmetric)
arginine (DMA) and N
-monomethylated
arginine (MMA) present in acid hydrolysates of lysed parent (RMT1) or mutant (rmt1) yeast cells after a 30-min in vivo labeling with [
H]AdoMet, as
described under ``Experimental Procedures.'' The samples were
mixed with 1 µmol of each of the non-isotopically labeled
standards, N
,N
-dimethylarginine
(asymmetric) and N
-monomethylarginine (both
obtained from Sigma). An equal volume of citrate sample dilution buffer
(0.2 M in Na
containing 2% thiodiglycol and
0.1% phenol at pH 2.2) was then added, and the resulting mixture was
loaded onto a high resolution amino acid analysis cation exchange
column. The column (Beckman AA-15 sulfonated polystyrene, 0.9 cm
diameter
11 cm height) was equilibrated with sodium citrate
buffer (0.35 M in Na
, pH 5.27) at 55 °C
and eluted at approximately 1 ml/min. After each run the column was
washed with 0.2 N NaOH for 20 min prior to the next run. One
minute fractions were collected, and [
H]
radioactivity was determined by counting a 700-µl aliquot of each
fraction (filled circles) by liquid scintillation in 5 ml of
fluor. An additional 100 µl of each fraction was analyzed for the
non-isotopically methylated amino acid standards using the ninhydrin
method (Gary and Clarke, 1995) (solid line). Briefly, the 100
µl column sample was diluted with 600 µl of water and mixed
with 300 µl of ninhydrin reagent (2% (w/v) ninhydrin and 3 mg/ml
hydrindantin in a solvent of 75% (v/v) dimethyl sulfoxide and 25% (v/v)
4 M lithium acetate at pH 4.2). The mixture was heated for 15
min at 100 °C, and the absorbance at 570 nm was measured. It should
be noted that the standard N
,N`
-dimethylarginine
(symmetric) always elutes between the positions of N
,N
-dimethylated (asymmetric)
arginine and N
-monomethylated arginine (see text
and Fig. 7B). The slightly earlier elution of the
[
H]methylarginine derivatives compared to their
cold standards is due to the change in molecular weight and pI of the
[
H]species versus the hydrogenated form
(Gottschling and Freese, 1962; Xie and Clarke, 1993). Panels B and D are enlargements of panels A and C, respectively, in the region where methylated arginines
elute.
Figure 6:
RMT1
is required for the asymmetric dimethylation of arginine residues in
cellular extracts. Panels A and C show the fraction
of asymmetrically dimethylated arginine (DMA) and
monomethylated arginine (MMA) formed during in vitro methylation reactions containing parent (RMT1) or mutant (rmt1) yeast extract with a 1-h incubation at 30 °C with
[H]AdoMet. The reactions contained either 269
µg of CH9100-2 extract or 338 µg of JDG9100-2 extract, buffer
(25 mM Tris-HCl, 1 mM sodium EDTA, and 1 mM sodium EGTA at pH 7.5), and 0.82 µM [
H]AdoMet (2.2 µCi) in a final volume of
34 µl. After 30 min, 15 µl of each reaction was removed and
placed in a glass vial (6
50 mm) and the protein was
precipitated by the addition of an equal volume of 25% (w/v)
trichloroacetic acid. The mixture was incubated at 25 °C for 10 min
before pelleting the precipitate at 4,000
g for 20 min
at 25 °C. The protein pellet was washed with -20 °C
acetone and dried before being acid hydrolyzed as described under
``Experimental Procedures.'' The hydrolyzed pellet was
resuspended in 50 µl of water, and 10 µl was loaded onto the
sulfonated polystyrene column as described in Fig. 5. Panels
B and D are enlargements of panels A and C, respectively, in the region where methylated arginines
elute.
Figure 7:
The
addition of the yeast GST-RMT1 or the mammalian GST-PRMT1 fusion
protein complements the methylation deficiency in rmt1 extract. Reactions (50 µl) in lanes 1-3,
contained 169 µg of rmt1 soluble extract protein, 0.7
µM [H]AdoMet (2.75 µCi), buffer
(25 mM Tris-HCl, 1 mM sodium EDTA, and 1 mM sodium EGTA at pH 7.5), and either 1.95 µg of GST-RMT1
protein, 2.0 µg of GST-PRMT1 protein (see ``Experimental
Procedures''), or no fusion protein, as the control. The reactions
were incubated and analyzed as described in Fig. 4A. In lanes 4 and 5, reactions (50 µl) contained 59
µg of soluble extract protein from RAT1 cells (see
``Experimental Procedures''), buffer, 0.7 µM [
H]AdoMet (2.75 µCi), with or without
the addition of 169 µg of extract protein from the rmt1 mutant cells. The reactions were incubated and analyzed as
described above. This panel shows the result of a 5-day exposure at
-80 °C. Molecular mass markers are indicated by the arrows.
The results of Fig. 5and Fig. 6provide conclusive evidence for a protein-arginine methyltransferase activity that is dependent on the RMT1 gene product. Furthermore, this activity represents the major arginine methyltransferase activity present in intact yeast cells. The analysis of in vivo methylated cells indicates that the RMT1 gene product is required for about 89% of the asymmetrically dimethylated arginine residues and about 66% of monomethylated arginine residues (Fig. 5, B and D). In vitro, the RMT1 gene product is responsible for 83% of the asymmetrically dimethylated arginine residues, but does not seem to be required for the production of monomethylarginine (Fig. 6, B and D). These results indicate the presence of at least one RMT1-independent protein-arginine methyltransferase in yeast.
The yeast polypeptides methylated when
incubated with the purified GST-RMT1 fusion protein (Fig. 7, lane 1) are mono- and asymmetrically dimethylated (Fig. 8A). The yeast GST-RMT1 fusion protein was
incubated with [H]AdoMet in the presence or
absence of rmt1 mutant extract. The proteins were then
precipitated and acid-hydrolyzed to quantitate the amount of
[
H]mono- and asymmetrically dimethylated arginine
residues formed (Fig. 8, A and C). In the
absence of rmt1 extract, only a small background of methylated
arginine residues was detected (Fig. 8, compare A with C).
Figure 8:
Polypeptides from the yeast rmt1 mutant extract are mono- and asymmetrically dimethylated on
arginine residues when incubated with the GST-RMT1 and GST-PRMT1 fusion
proteins. In panels A and B, the methylated
polypeptides seen in lanes 1 and 3 of Fig. 7were analyzed for mono- and asymmetrically dimethylated
arginine content. For panel A, 3.9 µg of GST-RMT1 protein
was incubated at 30 °C for 30 min with 253 µg of rmt1 extract protein, 0.82 µM [H]AdoMet (2.2 µCi), and buffer (25
mM Tris-HCl, 1 mM sodium EDTA, and 1 mM sodium EGTA at pH 7.5) in a final volume of 34 µl. For panel B, 1.55 µg of GST-PRMT1 was incubated at 30 °C
for 30 min with 338 µg of rmt1 extract protein, 0.97
µM [
H]AdoMet (2.2 µCi), and
buffer in a final volume of 29 µl. Both reactions were stopped by
the addition of an equal volume of 25% (w/v) trichloroacetic acid. The
samples were then acid-hydrolyzed and analyzed as in Fig. 5.
Corresponding control reactions (panels C and D) were
also prepared with only the fusion proteins,
[
H]AdoMet, and buffer. For the control reactions,
20 µg of bovine serum albumin was added to the reaction just prior
to trichloroacetic acid precipitation to obtain a higher recovery of
protein. All samples were co-chromatographed with 1 µmol of N
,N
-dimethylarginine
(asymmetric) (DMA) and 1 µmol of N
-monomethylarginine (MMA), shown as solid lines in the elution profile. In panel B, 1
µmol of N
,N`
-dimethylarginine
(symmetric) (DMA`) was also
included.
We also compared the ability of the yeast GST-RMT1 and
the mammalian GST-PRMT1 fusion proteins to cause the methylation of the
hypomethylated substrates present in the yeast rmt1 soluble
extracts. The purified GST fusion protein containing the rat PRMT1
sequence was able to efficiently methylate only a 55-kDa species, while
the corresponding yeast GST fusion protein promoted the methylation of
10 or more polypeptide species (Fig. 7, compare lanes 1 and 3). Thus the rat GST fusion enzyme appears to have a
quite restricted substrate specificity for yeast hypomethylated
proteins when compared to the yeast GST fusion protein. This narrow
specificity demonstrated by the rat fusion protein also reflects the
activity of the native rat enzyme, since the 55-kDa species is also the
major methylated polypeptide when a soluble extract of RAT1 cells is
used to methylate rmt1 mutant cytosol. We do find, however,
two additional minor methylated polypeptides at 34 and 24 kDa when rmt1 extract is incubated with RAT1 extract and
[H]AdoMet (Fig. 7). We determined that the
purified GST-PRMT1 fusion protein specifically mono- and asymmetrically
dimethylates the 55-kDa yeast substrate (Fig. 8, B and D).
We found that the purified GST-RMT1 fusion protein is able to methylate crude histones, recombinant hnRNP A1 (a gift from A. Krainer and A. Mayeda, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), and to a lesser extent cytochrome c and myoglobin (Fig. 9A). On the other hand, GST-RMT1 did not appear to methylate myelin basic protein (Fig. 9A). We found that hnRNP A1 is mono- and asymmetrically dimethylated on arginine residues by the GST-RMT1 fusion protein (Fig. 9B). We also determined that the chemical identity of the methylated residues in crude histones is mono- and asymmetric dimethylarginine (data not shown).
Figure 9:
The GST-RMT1 fusion protein methylates a
wide variety of substrates in vitro. In panel A,
purified GST-RMT1 protein (1.95 µg) was incubated with each of the
following: 100 µg of histones (calf thymus, Sigma; type IIAS), 490
ng of recombinant human hnRNP A1 protein, 100 µg of myelin basic
protein (bovine brain, Sigma), 100 µg of cytochrome c (horse heart, Sigma), and 100 µg of myoglobin (sperm whale
skeletal muscle, Sigma). The reaction also contained 0.93 µM [H]AdoMet (2.2 µCi) and buffer (25
mM Tris-HCl, 1 mM sodium EDTA, and 1 mM sodium EGTA at pH 7.5) in a final volume of 30 µl. The
reactions were incubated at 30 °C for 30 min and then stopped by
the addition of an equal volume of 2
SDS-gel electrophoresis
sample buffer. The samples were then loaded onto a 12.6% SDS-acrylamide
gel and fluorographed. This panel represents a 7-day exposure at
-80 °C. Arrowheads indicate the position of the
substrate proteins as determined by Coomassie stain. The arrows indicate the position of molecular mass standards described in Fig. 5. In order to determine the identity of the methylated
residue, reactions identical to those described in panel A (lanes 1 and 2) were incubated at 30 °C for
30 min. Bovine serum albumin (20 µg) was then added to each
reaction, and the entire mixture was transferred to a 6
50-mm
glass vial. An equal volume of 25% (w/v) trichloroacetic acid was
added, and the mixture was incubated at 25 °C for 10 min.
Precipitated protein pellets were acid-hydrolyzed and analyzed by
cation exchange chromotography as in Fig. 4. In panels B and C, the elution of [
H]
radioactivity is shown to coincide with the peaks of N
,N
-dimethylarginine
(asymmetric, DMA) and N
-monomethylarginine (MMA).
Figure 10:
The yeast and mammalian arginine
methyltransferase-GST fusions are differentially inhibited by S-adenosyl-L-homocysteine. rmt1 soluble extract (169
µg of protein) was incubated with 1.95 µg of GST-RMT1 protein
or 0.78 µg of GST-PRMT1 protein, 0.93 µM or 1.16
µM [H]AdoMet (2.2 µCi)
respectively, the indicated concentrations of S-adenosyl-L-homocysteine (SAH) and buffer
(25 mM Tris-HCl, pH 7.5, 1 mM sodium EDTA, and 1
mM sodium EGTA) in a final volume of 30 µl (panel
A) and 24 µl (panel B). The reactions were performed
at 30 °C for 30 min and were stopped by the addition of an equal
volume of 2
SDS-gel electrophoresis sample buffer. The samples
were then loaded onto a 10% SDS-acrylamide gel and fluorographed. Both
panels represent 4-day exposures at -80 °C. The arrows indicate the positions of molecular mass standards described in Fig. 4.
Although a number of proteins have been identified that contain methylated arginine residues and protein-arginine methyltransferase activities have been characterized in a variety of eukaryotic cells, the functional significance of this type of protein modification is not well understood (Kim et al., 1990; Lischwe, 1990; Clarke, 1993). For myelin basic protein, the only known protein that contains a symmetrically dimethylated arginine residue (Kim et al., 1990), methylation has been suggested to enhance the compaction of the opposing plasma membranes in myelin (Amur et al., 1986; Young et al., 1987; Rawal et al., 1992). For the group of proteins containing asymmetrically dimethylated arginine residues, a common thread has emerged; many of these species interact with RNA (Lischwe, 1990; Najbauer et al., 1993; Liu and Dreyfuss, 1995). Asymmetric dimethylation of arginine residues in these proteins could cause RNA binding to shift from a specific to a nonspecific mode due to the loss of specific hydrogen bonds (Calnan et al., 1991). Consistent with this model, there is reduced nucleic acid binding of methylated hnRNP A1 compared to the unmethylated form (Rajpurohit et al., 1994b). It is thus possible that arginine methylation modulates the activity of hnRNP A1 in pre-mRNA splicing or the activities of nucleolin and fibrillarin in processing preribosomal RNA (Lischwe et al., 1985; Aris and Blobel, 1991). Asymmetric dimethylation of arginine residues has also been proposed as a means of modulating nuclear localization of these and other proteins. For example, only the high molecular weight forms of basic fibroblast growth factor that contain methylated arginine residues are specifically found in the nucleus (Burgess et al., 1991). Additional roles of arginine methylation in the heat shock response (Desrosiers and Tanguay, 1988; Wang et al., 1992) and in virus-induced cell transformation (Enouf et al., 1979; Wang et al., 1992) have been suggested as well.
No
genes encoding a protein-arginine methyltransferase activity have been
previously identified. In this work, we show that the yeast ODP1 gene product, now designated RMT1, is a protein-arginine
methyltransferase that is similar to a rat gene product we have
recently characterized (Lin et al., 1996). The RMT1 protein
catalyzes the formation of N-monomethylarginine
and N
,N
-dimethyl-
(asymmetric) arginine residues on a number of endogenous yeast
substrates and on exogenous mammalian proteins, including those
containing a glycine and arginine-rich GAR domain.
In vivo analysis of methylated proteins in a rmt1 mutant strain indicates that the major activity responsible for asymmetrically dimethylating as well as monomethylating arginyl residues in S. cerevisiae is dependent upon a functional RMT1 gene product. Using soluble extracts from the parent and the rmt1 mutant yeast cells in in vitro assays, we confirmed that the predominant asymmetric dimethylarginine methyltransferase activity is dependent upon RMT1. However, the homogenization of the yeast cells appears to release an RMT1-independent monomethylarginine methyltransferase from a cellular compartment where the activity could not be detected in vivo. Direct evidence that RMT1 is a mono- and asymmetric dimethylarginine methyltransferase comes from the acid hydrolysis and amino acid analysis of in vitro reactions containing the GST-RMT1 fusion protein and recombinant hnRNPA1 methyl-accepting protein.
It should be noted that our results are in contrast to those of Liu and Dreyfuss(1995), where arginine methylation was not observed in yeast extracts when recombinant hnRNP A1 was used as a substrate. One possible explanation for this difference is that the yeast strains used here are protease-deficient strains and that the methyltransferase activity may be highly susceptible to proteolysis when cells are disrupted for extract preparation.
This RMT1-dependent methylation is clearly not essential for viability since the rmt1 cells grow similarly to the parent cells in YPD. Assays with purified substrates, using the GST-RMT1 fusion protein, suggest that this enzyme has a broad specificity for methyl-accepting substrates. The yeast fusion protein is not only able to methylate mammalian histones and recombinant hnRNP A1, but also cytochrome c and myoglobin. The latter two proteins have been characterized as substrates for a Euglena gracilis protein-arginine methyltransferase (Farooqui et al., 1985), and the former two proteins as substrates for the mammalian enzyme. The broad specificity of the yeast RMT1, indicated both by the ability of the fusion protein to methylate purified substrates and by the many substrate proteins present in hypomethylated rmt1 mutant extracts, suggests the wide-spread use of this post-translational modification in yeast. The comparatively restricted substrate specificity of the rat PRMT1 enzyme suggests the functions of a single yeast protein-arginine methyltransferase may be distributed among a family of related enzymes in higher eukaryotes, as is the case for other enzymes responsible for the posttranslational protein modifications (e.g. cyclin-dependent protein kinases; Grana and Reddy(1995)). In fact, analysis of the expressed sequence tag data base indicates that there appear to be at least two related human cDNA sequences that are highly similar to the rat PRMT/yeast RMT1 sequences.
Ghosh et al.(1988) partially purified two distinct protein-arginine methyltransferase activities from calf brain. One enzyme specifically methylated myelin basic protein (Ghosh et al., 1990), and the other was a histone arginine methyltransferase that was later shown to be more efficient toward hnRNP A1 (Rajpurohit et al., 1994a). Our substrate analysis of the yeast protein-arginine methyltransferase supports this idea of at least two distinct classes of arginine methyltransferases. The myelin basic protein-arginine methyltransferase specifically mono- and symmetrically dimethylates arginine residue 107 (Baldwin and Carnegie, 1971; Brostoff and Eylar, 1971), while the histone/hnRNP A1-specific enzyme mono- and asymmetrically dimethylates multiple arginine residues present in a GAR domain (Rajpurohit et al., 1992). The GST-RMT1 fusion protein methylated histones, hnRNP A1, cytochrome c, and myoglobin: substrates that can be modified to contain mono- or asymmetrically dimethylated arginine residues (Paik and Kim, 1969; Beyer et al., 1977; Karn et al., 1977; Farooqui et al., 1985). However, GST-RMT1 was not able to methylate myelin basic protein.
The potential in
vivo yeast arginine methyltransferase substrates identified by
Najbauer et al.(1993) have molecular sizes similar to those of
the methylated polypeptides observed when GST-RMT1 was incubated with
hypomethylated rmt1 cytosol and
[H]AdoMet. For example, the NSR1 polypeptide has
a predicted size of 45 kDa, but migrates as a 67-kDa polypeptide by
SDS-gel electrophoresis. NSR1 is an essential gene in yeast,
encoding a protein that not only binds nuclear localization sequences
(Lee et al., 1991) but also is involved in the processing of
pre-rRNA and is the homolog to the mammalian nucleolin (Kondo and
Inouye, 1992; Kondo et al., 1992). Another potential RMT1
substrate is NPL3, whose gene was isolated from a mutant defective in
localization of nuclear proteins and whose protein product migrates as
a 55-kDa species (Bossie et al., 1992). The RNA and
single-stranded DNA-binding protein SSB1 also contains a GAR domain and
migrates as a 45-kDa protein (Jong et al., 1987), while NOP1,
the essential yeast fibrillarin homolog, migrates as a 38-kDa protein
(Schimmang et al., 1989; Henriquez et al., 1990). The GAR1 gene was identified by Southern analysis with a cDNA
probe corresponding to the GAR domain of Xenopus fibrillarin.
The encoded protein migrates at 24.5 kDa by SDS-gel electrophoresis
(Girard et al., 1992). We plan to determine if these GAR
domain-containing proteins are in fact the major substrates in vivo and whether the lack of methylation affects their biological
activities. The hnRNP A1 methylation reaction is of interest because of
the availability of purified, recombinant protein as a substrate and
the recent partial purifications of a methyltransferase activity
capable of specifically methylating this substrate (Rawal et
al., 1994; Liu and Dreyfuss, 1995). Furthermore, the ability of
hnRNP A1 to preferentially promote splicing to more distal 5`-splice
sites has been well characterized (Mayeda and Krainer, 1992; Mayeda et al., 1993) and provides an assay for determining the
potential role of methylation in this context. The investigation into
the identity of other GST-RMT1 substrates will also be of great
importance in determining functions for arginine methylation.