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J. Biol. Chem., Vol. 275, Issue 22, 17122-17129, June 2, 2000
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From the
Received for publication, January 12, 2000, and in revised form, March 17, 2000
The Sm proteins B/B', D1, D2, D3, E, F, and G are
components of the small nuclear ribonucleoproteins U1, U2, U4/U6, and
U5 that are essential for the splicing of pre-mRNAs in eukaryotes. D1 and D3 are among the most common antigens recognized by anti-Sm autoantibodies, an autoantibody population found exclusively in patients afflicted with systemic lupus erythematosus. Here we demonstrate by protein sequencing and mass spectrometry that all arginines in the C-terminal arginine-glycine (RG) dipeptide repeats of
the human Sm proteins D1 and D3, isolated from HeLa small nuclear ribonucleoproteins, contain symmetrical dimethylarginines (sDMAs), a
posttranslational modification thus far only identified in the myelin basic protein. The further finding that human D1 individually overexpressed in baculovirus-infected insect cells contains
asymmetrical dimethylarginines suggests that the symmetrical
dimethylation of the RG repeats in D1 and D3 is dependent on the
assembly status of D1 and D3. In antibody binding studies, 10 of 11 anti-Sm patient sera tested, as well as the monoclonal antibody Y12,
reacted with a chemically synthesized C-terminal peptide of D1
containing sDMA, but not with peptides containing asymmetrically
modified or nonmodified arginines. These results thus demonstrate that
the sDMA-modified C terminus of D1 forms a major linear epitope for
anti-Sm autoantibodies and Y12 and further suggest that
posttranslational modifications of Sm proteins play a role in the
etiology of systemic lupus erythematosus.
The small nuclear ribonucleoproteins
(snRNPs)1 U1, U2, U5, and
U4/U6 are the major functional constituents of the spliceosome, a
nuclear multicomponent complex that catalyzes the splicing of nuclear
pre-mRNA in eukaryotes (1). The UsnRNPs consist of one (in the case
of U1, U2, and U5) or two (in the case of U4/U6) UsnRNAs and at least
50 distinct proteins (2). These proteins can be divided into two
groups: the Sm proteins B/B', D1, D2, D3, E, F, and G, found in each of
the UsnRNPs, and the specific proteins that are associated with only
one of the UsnRNP particles.
The Sm proteins play a key role in the biogenesis of the UsnRNPs. In
the cytoplasm, the Sm proteins form RNA-free hetero-oligomers E·F·G, D1·D2, and B/B'·D3 (3-6). After the nuclear export of the RNA polymerase II transcripts U1, U2, U4, and U5, the Sm protein complexes assemble on the Sm site, a conserved sequence that these UsnRNAs have in common (7). Interestingly, single Sm proteins do not
bind to the UsnRNA. Rather, the D1·D2 and E·F·G complexes first
bind to the Sm site, thus forming a so-called subcore snRNP intermediate. Subsequent association of the B/B'·D3 complex completes the formation of the core UsnRNP. The core UsnRNP is required for the
hypermethylation of the m7G cap of the U1, U2, U4, and U5
snRNAs to m3G caps (8, 9) and also constitutes part of the
bipartite nuclear localization signal necessary for the import of the
UsnRNPs into the nucleus (10, 11). Moreover, there is evidence that the
Sm proteins may stabilize the association of specific snRNP proteins
(12).
Sm proteins are not only of functional interest but also of clinical
relevance. Patients suffering from the autoimmune disease systemic
lupus erythematosus (SLE) spontaneously produce autoantibodies against
a multitude of cellular components (13-15). Antibodies against Sm
proteins (anti-Sm autoantibodies) are SLE-specific, in contrast to most
other SLE-associated autoantibody populations. Therefore, the
elucidation of the antigenic determinants recognized on the Sm
proteins is of significant diagnostic and immunopathological relevance.
In immunoblots, anti-Sm autoantibodies react predominantly with the Sm
proteins B/B', D1, D3, and, to a lesser extent, D2 (16, 17). The E, F,
and G proteins are also frequently recognized under native conditions,
such as in immunoprecipitation assays (18). The parallel recognition of
several Sm proteins by anti-Sm patient sera is not only due to distinct
antibody populations but also to cross-reactive epitopes on the Sm
proteins (16, 19), thus indicating that the Sm proteins share
structural homology. Indeed, all Sm proteins are evolutionarily
conserved in an approximately 70-90-amino-acid-long region designated
the Sm domain, which contains the Sm1 and Sm2 motifs (3, 20, 21). The
recent elucidation of the crystal structures of the B·D3 and D1·D2
complexes demonstrated that the fold of the Sm domain is also highly
conserved (22). Epitope mapping studies with the Sm proteins B/B' and
D1 have demonstrated that the Sm domains of these proteins indeed form discontinuous B-cell epitopes (23, 24). However, some putative linear
epitopes have also been mapped on the Sm proteins D1 and B/B'. These
epitopes are predominantly located in the C-terminal extensions of the
D1 and B/B' proteins and not in the Sm domain (25). The C terminus of
D1, for example, which contains a stretch of nine RG dipeptides (26),
was reported to form an epitope for anti-Sm autoantibodies (23,
27-30). However, in contrast to the native D1 protein, the C-terminal
epitope of D1 often reacted with autoantibodies from patients with
other diagnoses than SLE (30). Therefore, the disease-specific
recognition of the D1 C terminus by anti-Sm autoantibodies seems not
only to be sequence-dependent but could additionally depend
on other factors such as proper folding and/or posttranslational modifications.
C-terminal RG dipeptide repeats are found in D1 and D3 orthologs of
most organisms except yeast, suggesting that the RG stretches of D1 and
D3 play an important functional role in higher eukaryotes. RG-rich
regions are also found in several other nuclear proteins that are
involved in RNA processing (e.g. heterogeneous nuclear ribonucleoprotein A1, fibrillarin, nucleolin, and the yeast-RNA binding
protein Np/3p (for a review, see Ref. 31)). However, in contrast to D1
and D3, they contain RGG motifs. Interestingly, the
glycine/arginine-rich motifs of these proteins commonly contain asymmetrical dimethylarginines (aDMAs), a posttranslational
modification formed by type I protein arginine
N-methyltransferases (31). We surmised that methylation of
arginines in D1 could not only explain why synthetic RG-rich D1
peptides often react unspecifically (30) but also why human D1 protein
reacts only weakly and sometimes unspecifically with anti-Sm
autoantibodies when overexpressed in E. coli (32-34). We
therefore analyzed the Sm proteins D1 and D3 for posttranslational
modifications and investigated the influence of such modifications on
their antigenicity. Using protein sequencing and mass spectrometry, we
could demonstrate that the human Sm proteins D1 and D3 are dimethylated
in vivo in all nine and four positions, respectively, of
their C-terminal RG-stretches. In contrast to other nuclear proteins,
D1 and D3 contain symmetrical dimethylarginines (sDMAs), a
posttranslational modification thus far only identified in the myelin
basic protein (MBP). In epitope mapping studies, a synthetic C-terminal
peptide of D1 that contained sDMA reacted with 10 of 11 anti-Sm patient
sera and the monoclonal antibody Y12, while homologous peptides with
aDMA or nonmodified arginines were not recognized. Our data thus
demonstrate that the recognition of D1 by anti-Sm autoantibodies and
Y12 is strongly dependent on the symmetrical dimethylation of the C
terminus of D1.
Patient Sera--
Sm-positive patient sera (n = 11) were obtained from the Department of Rheumatology, Ghent University
Hospital (Ghent, Belgium). Selection of Sm-positive sera was based on
the use of the INNO-LIATM ANA (Innogenetics N.V., Ghent, Belgium), a
line immunoassay (LIA) containing natural SmD derived from purified
HeLa UsnRNPs. Control sera tested negative for anti-nuclear factor in
indirect immunofluorescence on Hep2 cells and showed no reactivity on
the INNO-LIATM ANA.
Isolation of Sm Proteins and Protein
Sequencing--
Purification of HeLa UsnRNPs and subsequent separation
of Sm proteins by SDS-polyacrylamide gel electrophoresis have been described earlier (16). Coomassie Blue-stained bands of the respective
Sm proteins were excised and directly submitted to proteolytic
digestion according to Rosenfeld et al. (35). Briefly, the
excised gel slices were washed twice with 2 ml of water for 1 h
and then shaken strongly in an aqueous solution (pH 6.4) of 40%
acetone, 10% triethylamine, and 5% acetic acid for 2 h. After washing three times for 1 h with 2 ml of water, the gel slices were dried and proteolytically digested with 0.5-2.0 µg of
endoproteinase Lys-C (Roche Molecular Biochemicals) in 200-500 µl of
digestion buffer (100 mM Tris-HCl, pH 8.0, 10%
acetonitrile, 1 mM K3EDTA). The peptide mixture
was separated by HPLC on a C4 column (Vydac, Hesperia, CA) using a
10-70% gradient of solvent B (70% acetonitrile, 0.1%
trifluoroacetic acid). Manually recovered peptide fractions were then
sequenced by Edman degradation using a Procise 492 sequencer equipped
with an on-line 140C phenylthiohydantoin analyzer (Perkin-Elmer). To
distinguish methylated arginines from nonmethylated arginines, we first
applied a combination of either monomethylarginine (MMA) and sDMA or
MMA and aDMA to a biobrene-coated, trifluoroacetic acid-treated glass
fiber filter (Perkin-Elmer) and performed an Edman degradation cycle
for each combination. Next, the human C-terminal D1 peptide, the
C-terminal D3 peptide, or the baculo-derived C-terminal peptide was
applied on the same filter and sequenced to completion. All additional
standards were purchased from Sigma.
Overexpression and Purification of D1 in Escherichia coli and
Baculovirus-infected Insect Cells--
A cDNA containing the
sequence of human D1 (26) was inserted as a
BamHI/XbaI fragment into the expression vector
pIGFH111 (Innogenetics), thus introducing the first 25 amino acids of
mouse tumor necrosis factor and six histidines to the amino terminus of
D1. After transfection into the E. coli strain SG4044
(pc1857) and temperature induction, a strong protein band in the
expected range of 18 kDa could be detected in the soluble fraction.
This protein was verified to be recombinant D1 by immunoblotting with an anti-tumor necrosis factor antibody (Innogenetics). After lysing the
cells by French pressing in lysis buffer (10 mM Tris-HCl, pH 6.8, 100 mM KCl, 25 mM
For overexpression of D1 in baculovirus-infected insect cells, a
520-base pair DraI-SbaI fragment of
pIGFH111hSmD1 (see above) was inserted into a
BamHI-XbaI-digested baculovirus transfer plasmid pVL1393 (Pharmingen, San Diego, CA) in which the fusion gene is under
transcriptional control of the strong baculovirus polyhedrin promotor.
After transfection of the vector (as described by the manufacturer), a
D1 protein was expressed, as evidenced by immunoblotting (see above).
After lysis, baculo-derived D1 was purified on a nickel column as
described above.
Mass Spectrometry--
Matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of
the C-terminal D1 peptide was performed on a Voyager-DE STR (PerSeptive
Biosystems, Framingham, MA) mass spectrometer equipped with delayed
extraction and operated at an accelerating voltage of 20 kV and 150-ns
delay. Desorption and ionization of the sample was carried out with a
nitrogen laser (337 nm). Samples were applied on a stainless steel
sample plate using the thin layer method (36) and measured in
reflectron mode. MALDI-TOF-obtained spectra were externally calibrated
using a standard peptide mixture of angiotensin I 1296.6853 [M + H]+, ACTH-(1-17) 2093.0867 [M + H]+,
ACTH-(18-39) 2465.1989 [M + H]+, and ACTH-(7-38)
3657.9294 [M + H]+. The observed mass peaks were
annotated manually. Electrospray mass spectrometry of recombinant D1
was performed in a Bio-Q-quadrupole mass spectrometer equipped with an
electrospray ion source (Micromass, Wytenshawe, United Kingdom). For
analysis, 20 pmol of protein or peptide were applied in 10 µl of 50%
acetonitrile, 1% acetic acid. Calibration was performed with 50 pmol
of horse heart myoglobin.
Peptide Synthesis and LIA--
Peptides were synthesized on
Tentagel S resin (Rapp Polymere GmbH, Germany) using a Rainin Symphony
Multiplex synthesizer (Protein Technologies, Tucson, AZ) with standard
Fmoc chemistry. Standard double couplings were performed by using a
4-fold excess of Fmoc-protected amino acids activated in
situ with equimolar amounts of N-hydroxybenzotriazole
and 2-(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium tetrafluoroborate twice for 20 min each time. Fmoc-protected
asymmetrical and symmetrical dimethylarginine (Bachem, Switzerland)
were incorporated similarly. After completion of the peptide synthesis,
the peptide was cleaved from the resin by incubation for 2.5 h
with a mixture of 90% trifluoroacetic acid, 5% thioanisole, 3%
ethanedithiol, and 2% anisole. The peptide was precipitated from the
mixture using t-butyl methyl ether. After centrifugation,
the pellet was washed three times with t-butyl methyl ether
and dried overnight in a vacuum. The purity of the crude peptide was
checked by reversed-phase HPLC.
For LIA, purified, gel-eluted HeLa proteins, from which SDS and
Coomassie were removed by ion pair extraction (16) or purified recombinant proteins were sprayed as lines on a nylon membrane with a
plastic backing and treated as described previously (37). Patient sera
and Y12 were applied in a dilution of 1:200. A 3-fold molar excess of
biotinylated synthetic peptides in 50 mM sodium carbonate
buffer (pH 9.6) were complexed to streptavidin by incubating for 1 h at 37 °C, before applying to the membrane. In order to visualize
the applied proteins or peptides, a nonblocked LIA strip was stained
with colloidal gold (Aurodye; Amersham Pharmacia Biotech) according to
the manufacturer's procedure.
The Human Sm Proteins D1 and D3 Contain sDMAs at Their Carboxyl
Termini--
To investigate whether the RG-rich stretches of the human
D1 and D3 proteins contain methylated arginines, we performed protein sequencing and mass spectrometry. HeLa UsnRNPs were initially purified
by immunoaffinity chromatography with an H2O antibody directed against
the m3G cap of UsnRNPs (38), and the protein constituents
of the human UsnRNPs were then fractionated by High-TEMED SDS-polyacrylamide gel electrophoresis (16). Subsequently, those bands
containing the D1 and D3 proteins were excised from the gel and
proteolytically digested with endo-Lys-C. This procedure resulted in a
C-terminal 29-amino acid-long D1 fragment (amino acids 91-119) and a
23-amino acid-long D3 fragment (amino acids 105-124) (see Fig.
1). After fractionation of the D1 and D3
peptide mixtures by HPLC, we were able to isolate both C-terminal
peptides, so that all C-terminal RG dipeptides of D1 and D3 were
analyzed (Fig. 1).
To initially determine whether the C-terminal peptide of D1 contains
posttranslational modifications, we performed mass spectrometry (MALDI-TOF). A protonated major mass peak, isotopically resolved at
3366.68 Da, was detected (Fig. 2). The
observed value clearly deviated from the theoretical mass expected for
a nonmodified D1-(91-119) peptide. Moreover, the mass difference fit
almost perfectly for a D1 peptide with dimethylated arginines at all positions of its RG-rich stretch (the theoretical protonated
monoisotopic value of D1-(91-119) with nine DMAs is 3366.10 Da). A
minor peak of 3238.58 Da, obtained to a lesser extent after
proteolytical digestion, was also observed and is consistent with a
dimethylated D1-(92-119) peptide (Fig. 2). Two additional peaks of
3252.85 and 3380.65 Da corresponded to D1-(92-119) and D1-(91-119)
peptides with an additional methyl group, due to esterification of the carboxyl termini upon storage in an acidified methanolic solution. The
MALDI-TOF analysis of D1 thus not only demonstrated the presence of
posttranslational modifications in the C terminus of D1, but also
strongly suggested the presence of DMAs.
To confirm the results of our mass spectrometry experiments and to
distinguish between asymmetrically and symmetrically dimethylated arginines (see Fig. 3), we analyzed the
C-terminal D1 and D3 peptides by protein sequencing. For this purpose,
we not only used the normal PTH-derivative standard during the
sequencing run but additionally applied MMA, aDMA, and sDMA as a
standard for the HPLC-based identification of amino acids. Fig.
3A shows an alignment of the Edman degradation cycles of the
gel-purified C-terminal peptide of HeLa D1 with the HPLC elution
profiles of PTH-modified MMA, aDMA, and sDMA (indicated by
arrows). We obtained a clear separation of arginine, MMA,
aDMA, and sDMA (Fig. 3, A and B). When we
compared the elution profiles of the C-terminal peptide of HeLa D1 with
the applied standards, we could reproducibly identify symmetrical DMAs
in the C terminus of D1. As emphasized by the cycle numbers in Fig. 3A (only the cycles corresponding to the arginines are
shown), sDMA is present at all nine positions in the RG-rich stretch of D1. Only the arginines at positions 2, 28, and 29 of the D1 peptide (positions 92, 118, and 119 of D1), which are not flanked by glycines, were not dimethylated (data not shown), thus providing evidence that
the methyltransferase modifying D1 recognizes solely its RG repeat. In
agreement with our mass spectrometry experiments (Fig. 2), we could not
observe even minor peaks corresponding to arginine or MMA (see Fig.
3A). The absence of detectable amounts of undermethylated D1
precursors in HeLa nuclear extracts may hint at a cytoplasmic
localization of the D1-modifying methyltransferase.
In the D3-derived peptide encompassing amino acid residues 105-124,
four symmetrically methylated arginines at positions 110, 112, 114, and
118 of the D3 protein were identified (data not shown). Interestingly,
the sDMA at position 110 of D3, in contrast to all other modified
arginines in the RG-rich stretches of D1 and D3, was not preceded by a
glycine (alanine instead of glycine; see Fig. 1). This raised the
possibility that such RG dipeptides generally could serve as substrates
for posttranslational methylation. However, RG dipeptides present in
the Sm motif 2 of D1 and D3 (amino acid 61 in D1 and 64 in D3; Fig. 1)
were not modified. Also, methylated arginines were not found in the
single RG dipeptides present in the Sm proteins E, F, G, and D2 (data
not shown). As observed with D1, the sDMA precursors arginine and MMA
were not detected in D3. These results thus demonstrated that, in
contrast to several RGG box-containing proteins that contain
asymmetrical DMA, the human Sm proteins D1 and D3 are symmetrically
dimethylated. Moreover, since we isolated the D1 and D3 proteins from
biochemically purified HeLa UsnRNPs, symmetrical dimethylation of D1
and D3 occurs in vivo.
Recombinant D1 from E. coli and Baculovirus-infected Insect Cells
Is Only Poorly Recognized by Anti-Sm Patient Sera in LIAs--
D1
overexpressed in E. coli has been reported to react only
weakly and sometimes unspecifically with patient sera (see
Introduction). Moreover, the prototypical anti-Sm antibody, Y12 (39),
strongly reacted with native D1, D3, and B/B' in immunoblots but not
with recombinant D1 overexpressed in E. coli (Ref. 16 and
data not shown). It was thus conceivable that the B-cell epitopes
recognized by Y12, and by at least some anti-Sm autoantibody
populations, require sDMA for efficient recognition. To investigate
this hypothesis, we overexpressed D1 in either E. coli or in
baculovirus-infected insect cells. For one-step purification by nickel
affinity chromatography, both proteins were overexpressed as fusion
proteins containing the first 25 amino acids of mouse tumor necrosis
factor and six histidines (see "Experimental Procedures"). In
electrospray mass spectrometry, we observed a mass of 17,435 Da for the
E. coli-derived D1, which was the expected mass for the
full-size fusion protein without any posttranslational modifications.
In contrast, the purified D1 from the baculovirus expression system
yielded several peaks with significantly higher masses (data not
shown), thus providing evidence that posttranslational methylation of
recombinant D1 had taken place. To further investigate the
dimethylation status of the baculo-derived D1, we performed protein
sequencing as described for the HeLa-derived D1 protein (see Fig.
3B). Although we could isolate a minor HPLC fraction
containing nonmodified peptide D1-(91-119), the majority of the
baculo-derived D1 was indeed dimethylated, as predicted by the mass
spectrometry (see above). However, in contrast to the HeLa-derived D1
protein, the baculo-derived protein contained asymmetrical, as opposed
to symmetrical, dimethylarginines (see Fig. 3B). The less
prominent peaks preceding the aDMAs in the HPLC elution profiles in
Fig. 3B co-eluted with the dimethylation precursors,
arginine and MMA.
To analyze the immunological properties of the recombinant D1 proteins,
we applied equal amounts of baculo-derived D1, E. coli-derived D1, and a mixture of gel-purified human D1, D2, and D3 (HeLa D1/D2/D3 in Fig. 4A),
which served as a positive control, to nylon strips. The strips were
then incubated with anti-Sm sera from SLE patients and the monoclonal
anti-Sm antibody Y12. As negative controls, we used sera from patients
with mixed connective tissue disease, containing high titers of
anti-RNP autoantibodies against the U1-A, U1-C, and U1-70K proteins
(RNP-positive in Fig. 4) as well as sera from healthy patients that did
not contain antinuclear autoantibodies (ANA-negative in Fig. 4). While
a mixture of D1, D2, and D3 from HeLa cells reacted significantly with
all of the tested anti-Sm patient sera, the E. coli-derived
D1 gave only weak signals, which were in the intensity range of signals obtained with control sera (compare D-positive sera with ANA-negative and RNP-positive sera in Fig. 4A). Interestingly, also the
baculo-derived D1 reacted only weakly with anti-Sm patient sera; only
four bands of significant intensity were observed with baculo-derived
D1 (sera Sm56, Sm83, Sm111, and Sm130 in Fig. 4A). Note that
similar amounts of antigen had been applied to the strips, as
controlled by aurodye staining (Fig. 4A, lane
1). To verify that the tested anti-Sm patient sera contained
anti-D1 autoantibodies, we additionally tested the patient sera with
single HeLa-derived D1, D2, and D3 proteins in LIA (Fig.
4B). Except for serum Sm124, which reacted exclusively with
D2 (Fig. 4B, lane 3), all anti-Sm sera
reacted significantly with HeLa-derived D1, including those that did
not react with baculo-derived D1 (compare, for example, Sm34;
lane 5 in Fig. 4, A and B).
Although we cannot fully exclude the possibility that the weak
antigenicity of the recombinant D1 proteins was due to the extra amino
acids of the fusion region, the less frequent recognition of both
E. coli-derived and baculo-derived D1 indicated that the
presence of sDMAs strongly increases the antigenicity of the D1
protein.
Anti-Sm Patient Sera Specifically Recognize the sDMA-containing
Carboxyl Terminus of D1--
The predominant recognition of
HeLa-derived D1 in LIA strongly suggested that the symmetrically
dimethylated C terminus of D1 forms a B-cell epitope for anti-Sm
autoantibodies. To analyze this, we chemically synthesized peptides
containing nine aDMAs, nine sDMAs, or nine arginines in the in
vivo methylated positions and used them as antigens in LIA
experiments with the aforementioned patient sera. All peptides were
purified by HPLC and characterized by mass spectrometry. The presence
of sDMA was demonstrated by sequencing. To ensure efficient binding of
the peptides to the membrane, we conjugated them with an N-terminal
biotin residue during peptide synthesis and formed a
streptavidin-peptide complex before applying the antigens to the
membrane. Indeed, the complexed peptides bound well to the membrane
(Fig. 4C, lane 1). As shown in Fig.
4C, 10 of 11 anti-Sm patient sera significantly reacted with
the sDMA-containing peptide. In contrast, peptides with either arginine
or aDMA were not recognized by anti-Sm patient sera. These data not
only demonstrate that anti-Sm autoantibody populations frequently
recognize the C terminus of D1 directly but also show that the
efficient recognition of this epitope is dependent on the presence of
sDMA. Since sera from mixed connective tissue disease patients with
high titers of anti-RNP autoantibodies and control sera from healthy
patients did not react with the sDMA-containing peptide, the reaction
was SLE-specific. The specificity of the LIA was further substantiated
by the finding that the only anti-Sm patient serum that did not react
with the peptide was a serum that exclusively reacted with D2 (see Fig.
4, B and C, lane 3). The
negative signal observed with this serum was thus due to the absence of
anti-D1 autoantibodies. Strikingly, the monoclonal antibody Y12 also
reacted with the peptide D1-(95-119) sDMA, but not with D1-(95-119)
Arg or D1-(95-119) aDMA (Fig. 4C, lane
13). Previous studies using synthetic peptides or E. coli-derived deletion mutants had always failed to generate
significant signals with this antibody (see "Discussion"). Our
results indicate that Y12 also requires sDMA for antigen recognition,
at least in the epitope localized on D1. Moreover, since an
sDMA-containing RG-rich stretch is also present in the D3 protein, an
epitope in this region can easily explain the cross-reactivity of Y12
and anti-Sm autoantibodies with D1 and D3.
In this paper, we demonstrate by protein sequencing and mass
spectrometry that the human Sm proteins D1 and D3 are symmetrically dimethyated at C-terminal arginines in vivo. This is the
first time that posttranslational modifications have been localized in
Sm proteins. The presence of the unusual amino acid sDMA in D1 and D3
could have implications both for snRNP biogenesis and SLE etiology.
The elucidation of the antigenic determinants recognized by anti-Sm
autoantibodies is of considerable interest to understand and diagnose
the autoimmune disease SLE (see Introduction). In antibody binding
studies, we could clearly identify a linear epitope for anti-Sm
autoantibodies and the monoclonal antibody Y12 on the amino acids
95-119 of the human D1 protein. Importantly, the intensities of the
signals observed with the D1 peptide 95-119 containing sDMA were in
the range of the signals obtained with the native D1 protein (Fig. 4,
compare B and C). Previous studies have also
mapped an epitope for anti-Sm autoantibodies at the C terminus of D1
(23, 27-29, 40). However, in these studies, the analyzed D1 fragments
did not contain sDMA and were not compared with a wild type protein,
raising the possibility that the binding of anti-Sm autoantibodies was
only weak. Indeed, in our hands, a nonmodified D1 peptide 95-119 and
an aDMA-containing peptide 95-119 showed only basal level signal
intensities, as compared with the native D1 protein (Fig. 4). Our data
thus demonstrate that the presence of sDMA strongly increases the
affinity of anti-Sm autoantibodies for the C terminus of D1.
The sDMA-containing peptide D1-(95-119) is the first synthetic peptide
found to significantly react with the prototypical anti-Sm antibody Y12
(Fig. 4C). Deletion mutagenesis, using in vitro
translated proteins, has located the Y12 epitope to the C termini of
the Sm proteins D1 and B/B' (24, 41) and the ribosomal S10 protein
(42), all of which contain at least one GRG tripeptide. However,
neither synthetic peptides encompassing the mapped epitopes nor
E. coli-derived D1, D3, or B/B' reacted significantly with
Y12 (24).2 Our data therefore
suggest that symmetrically dimethylated RG stretches constitute a major
Y12 epitope. The dimethylation status of B/B' or S10 is not yet clear;
however, the presence of GRG tripeptides in the mapped Y12 epitopes of
these proteins raises the possibility that they may also contain sDMA.
However, although protein sequencing of single RG dipeptides in the E,
F, and G proteins revealed that these proteins are not dimethylated
(data not shown), an E·F·G complex is significantly
immunoprecipitated by Y12 (18). This indicates that Y12 may recognize
more than one epitope on the Sm proteins.
The high efficiency by which the sDMA-containing peptide is recognized
by anti-Sm autoantibodies and Y12 demonstrates that the C-terminal,
linear epitope contributes significantly to the antigenicity of the
full-length human D1 protein. Accordingly, the E. coli-derived D1, which contained nonmodified arginines was not
recognized by anti-Sm autoantibodies (Fig. 4, compare A and
C). However, 4 of 11 anti-Sm sera significantly reacted with
the aDMA-containing baculo-derived D1 but not with the aDMA-containing peptide. This indicates that the baculo-derived D1 contains additional epitopes, e.g. discontinuous epitopes on the Sm domain (see
Introduction), that were recognized by anti-Sm autoantibodies. Ou
et al. (32) reported that 96% of anti-Sm patient sera
recognized recombinant D1 overexpressed in baculovirus-infected insect
cells in enzyme-linked immunosorbent assays, while 71% of patient sera
recognized D1 overexpressed in E. coli. The increased
antigenicity of the baculo-derived D1 as compared with the E. coli-derived D1 suggests that the asymmetrical dimethylation of D1
in the former case stabilizes conformational epitopes on the Sm domain.
The same may be true for the symmetrical dimethylarginines. The lower
frequency of recognition of the recombinant D1 proteins in our LIA as
compared with the study of Ou et al. could therefore be due
to the increased degree of renaturation in the enzyme-linked
immunosorbent assay protocol used by Ou et al. (Ref. 32; see
also Ref. 40). However, since both the HeLa-derived D proteins and the
peptide D1-(95-119) sDMA were recognized with a high frequency (10/11)
and specificity by patient sera in our study, native Sm proteins and
the sDMA-containing peptide are interesting substrates for the
diagnosis of SLE. Studies to investigate the specificity and
sensitivity of anti-Sm autoantibody detection in an extended SLE
patient population, using the peptide D1-(95-119) sDMA as an antigen,
are currently under way.
Little is known about the etiology of SLE and other autoimmune
diseases. We have demonstrated that symmetrical dimethylation can
increase the affinity of anti-Sm autoantibodies toward their antigenic
target. Similarly, a class of rheumatoid arthritis-associated autoantibodies (APF/AKA) reacted exclusively with citrulline-containing peptides of the rheumatoid arthritis autoantigen filaggrin but not with
homologous arginine-containing peptides (43, 44). Autoantigens
containing asymmetrically dimethylated arginines have also been
described: fibrillarin in scleroderma (45), heterogeneous nuclear
ribonucleoprotein A1 in connective tissue diseases (46), and nucleolin
in SLE (47). It is therefore tempting to speculate that
posttranslational modifications might play a role in the etiology of
SLE and other autoimmune diseases. Recently, much attention has been
paid to posttranslational protein modifications during apoptotic
processes, which may lead to the subsequent development of
autoantibodies (48). Abnormal apoptosis, such as observed in SLE (for a
review, see Ref. 49), could result in the continuous presentation of
modified autoantigens that are normally retained in the nucleus (50).
During apoptosis, nuclear, dimethylated D1 could induce an autoimmune
response in an antigen-driven manner. To investigate this hypothesis,
it will be of considerable interest to analyze the immunogenicity of
the dimethylated C terminus of D1 in animal models.
The D1 and D3 proteins are the first examples of human nuclear proteins
that are symmetrically dimethylated in vivo. Other dimethylated nuclear proteins contain asymmetrical DMAs (see
Introduction). Indeed, the only other human protein shown to contain
sDMA to date is MBP, which is a major component of the nerve sheath and implicated in the autoimmune disease multiple sclerosis (51). This
raises the question of which protein arginine
N-methyltransferase is responsible for the methylation of
the D1 and D3 proteins. Type I methyltransferase, which was recently
cloned from yeast (52), rat (53) and humans (54), catalyzes the
formation of aDMAs in histones and heterogeneous nuclear
ribonucleoprotein A1 (for a review, see Ref. 31). In contrast, the gene
for the type II methyltransferase that specifically modifies MBP to
create sDMAs has not yet been identified. The symmetrical dimethylation of D1 and D3 would suggest that these proteins are substrates for a
type II methyltransferase. MBP contains only a single sDMA at position
107 (51), and the amino acid sequence surrounding Arg-107
(104GKGRGL109) slightly resembles the D1 RG
repeat. Interestingly, a heptapeptide with alternating glycines and
arginines (GRGRGRG), such as found in D1, was the most effective methyl
acceptor for a partially purified MBP-specific type II protein
methyltransferase (55). However, only MMA with a minor fraction of aDMA
was formed in vitro with this peptide, providing evidence
that methyltransferases may change their activity dependent on the
substrate. It is therefore possible that proper dimethylation of D1 and
D3 requires a particular hetero-oligomeric protein environment,
e.g. assembled core UsnRNPs or Sm protein complexes (see
Introduction). Proper assembly as a prerequisite for proper
dimethylation could easily explain our observation that baculo-derived
D1 contained aDMA instead of sDMA (see Fig. 3B), since the
overexpressed D1 protein does not come in contact with stoichiometric
amounts of the other Sm proteins or UsnRNA to form the D1·D2 complex
or core UsnRNPs. To unambiguously identify and characterize the
D1/D3-modifying enzyme, detailed in vitro methylation
studies with recombinant and/or purified methyltransferases and with
different substrates (such as the heteromeric protein complexes
involved in Sm core formation) will be necessary.
The biological function of arginine methylation is largely unknown. The
strongly increased binding of Y12 and anti-Sm autoantibodies to D1 in
the presence of sDMA suggests that the methylation of the D1 and D3
proteins may modulate interactions with other cellular components. In
the case of MBP, the sDMA at position 107 is indeed involved in the
integrity and compactness of myelin. Moreover, it has been demonstrated
that the MBP-specific methylase activity occurs concurrently with the
myelination process (for a review, see Ref. 56). Likewise, the
methylation of D1 and D3 could stabilize or weaken protein-protein or
protein-RNA interactions, thus regulating the biogenesis of UsnRNPs.
The formation of complexes between the Sm proteins (see Introduction)
is probably not affected by arginine methylation, since the yeast D1
and D3 homologues, which form equivalent complexes, lack an RG-rich
stretch (57, 58). In addition, it has been demonstrated for the human
Sm proteins that the Sm domain alone is sufficient for complex
formation (3, 22). However, arginine methylation could be involved in
the binding of the Sm core to UsnRNP-specific proteins (12) or could reduce unspecific protein-RNA interactions of D1 and D3 by steric hindrance (59). It has also been suggested that methylated arginines may facilitate transport events (60). Since one part of the bipartite
nuclear localization signal of the UsnRNPs is situated on the core
proteins (10, 11), it is possible that arginine methylation facilitates
the binding of the properly assembled core-UsnRNP to a still
unidentified nuclear import factor. Consistent with this possibility,
our data suggest that methylation of D1 takes place in the cytoplasm,
since all D1 molecules we isolated from nuclear extracts were
completely dimethylated. If methylation were to occur in the nucleus,
minor amounts of unmodified arginine and MMA would be expected at
positions 98-114 of D1. To understand the biological function of the
symmetrical dimethylation of the D1 and D3 proteins, it will therefore
be of considerable interest to analyze the cytoplasmic assembly
intermediates of the UsnRNPs for dimethylated arginines.
We thank Inge Vanneste, Caroline Dobbels,
Greet Hendrickx, Martine Dauwe, Carla Missiaen, and Irene Öchsner
for skillful technical assistance; the Departments of Protein
Expression, Purification, and Peptide Synthesis of Innogenetics for
valuable contributions; and Fred and Claire Shapiro for editorial
support. We also thank Cindy L. Will and Veronica A. Raker for critical
reading of the manuscript and helpful discussions.
*
This work was supported in part by a grant from the Deutsche
Forschungsgemeinschaft (to R. L.).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.
**
To whom correspondence and reprint requests may be addressed: Lydie
Meheus, Innogenetics N.V., Industriepark Zwijnaarde 7, Box 4, B-9052
Ghent, Belgium. Tel.: 32-9-2410711; Fax: 32-9-2410907; E-mail:
Lydie_Meheus@innogenetics.com or Reinhard Luhrmann, Tel.: 49-551-2011407; Fax: 49-551-2011197; E-mail:
R.Luehrmann@gwdg.de.
2
H. Brahms and R. Lührmann, unpublished observations.
The abbreviations used are:
snRNP, small nuclear
ribonucleoprotein;
SLE, systemic lupus erythematosus;
sDMA, symmetrical
dimethylarginine;
aDMA, asymmetrical dimethylarginine;
MMA, monomethylarginine;
MALDI-TOF, matrix-assisted laser
desorption/ionization time-of-flight;
LIA, line immunoassay;
MBP, myelin basic protein;
Fmoc, N-(9-fluorenyl)methoxycarbonyl;
PTH, phenylthiohydantoin;
HPLC, high pressure liquid chromatography;
ACTH, adrenocorticotropic hormone;
UsnRNA, uridyl-rich small nuclear
RNA;
UsnRNP, uridyl-rich small nuclear ribonucleoprotein.
The C-terminal RG Dipeptide Repeats of the Spliceosomal Sm
Proteins D1 and D3 Contain Symmetrical Dimethylarginines, Which Form a
Major B-cell Epitope for Anti-Sm Autoantibodies*
§,
,§**
Institut für Molekularbiologie und
Tumorforschung, Emil-Mannkopff-Str. 2, D-35037 Marburg, Germany, the
§ Max-Planck-Institute of Biophysical Chemistry, Department
of Cellular Biochemistry, Am Faßberg 11, D-37070 Göttingen,
Germany, ¶ Innogenetics N.V., Industriepark Zwijnaarde 7, Box 4, B-9052 Ghent, Belgium, and the Department of Rheumatology, Ghent
University Hospital, B-9000 Ghent, Belgium
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminocaproic
acid, 1 mM dithiothreitol, 2 mM
phenylmethylsulfonyl fluoride) and centrifugation at 4 °C (20 min at
27,000 × g), guanidinium HCl was added to the
supernatant to a final concentration of 4.5 M, and
recombinant protein was purified by affinity chromatography on a nickel
column (nickel-Sepharose; Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Location of sDMAs in the amino acid sequences
of the Sm proteins D1 and D3. The RG stretches are presented in
boldface type, and sDMAs are indicated by
asterisks. The position of the Sm 1 and Sm 2 motifs is shown
by the consensus sequence below the protein sequences.
Sequenced C-terminal D1 and D3 fragments obtained by proteolytic
digestion with endo-Lys-C are indicated by arrows.
View larger version (18K):
[in a new window]
Fig. 2.
MALDI-TOF mass spectrum of an HPLC-purified
fraction containing the modified arginine C-terminal peptide that was
obtained by endo-Lys-C digestion of human D1.
View larger version (58K):
[in a new window]
Fig. 3.
A, PTH-derivative sequence analysis of
an HPLC-purified fraction containing the C-terminal peptide generated
by endo-Lys-C digestion of human D1. An alignment is shown of the Edman
degradation cycles 8, 10, 12, 14, 16, 18, 20, 22, and 24 (corresponding
to arginines in positions 98, 100, 102, 104, 106, 108, 110, 112, and
114 of D1). The elution profiles of MMA, aDMA, and sDMA are indicated
by arrows. The structure of sDMA is shown on the
right. B, PTH-derivative sequence analysis of an
HPLC-purified fraction containing the C-terminal peptide generated by
endo-Lys-C digestion of baculo-derived D1. The same Edman degradation
cycles as in A are aligned. The structure of aDMA is shown
on the right.
View larger version (74K):
[in a new window]
Fig. 4.
LIA with patient sera and different Sm
antigen sources. The strips were tested with 11 anti-Sm patient
sera (SmD-positive), five anti-RNP sera from patients with
mixed connective tissue disease (RNP-positive), and five
control sera that were found negative for anti-nuclear antibodies
(ANA-negative). The designations of the sera are indicated
at the top. The antigen sources are indicated to the
left. The amount of applied antigen is indicated to the
right. A, baculo-derived D1, E. coli-derived D1, and a mixture of gel-purified human D1, D2, and
D3 from HeLa cells were applied to the strips at a concentration of
7.5, 15, and 30 ng/line. B, 15 ng of gel-purified, human D1,
D2, or D3 protein from HeLa cells were applied as antigen source.
C, line immunoassay with synthetic peptides, encompassing
the D1 amino acids 95-119. Peptide D1-(95-119) Arg contained
nonmodified arginines instead of sDMA in the in vivo
dimethylated positions. Peptide D1-(95-119) aDMA contained aDMA
instead of sDMA. Peptide D1-(95-119) sDMA contained sDMA in the
in vivo dimethylated positions. All peptides were applied to
the membrane at a concentration of 15 ng/line.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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 G. B. Gonsalvez, L. Tian, J. K. Ospina, F.-M. Boisvert, A. I. Lamond, and A. G. Matera Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins J. Cell Biol., August 27, 2007; 178(5): 733 - 740. [Abstract] [Full Text] [PDF]
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 J.-L. Liu and J. G. Gall U bodies are cytoplasmic structures that contain uridine-rich small nuclear ribonucleoproteins and associate with P bodies PNAS, July 10, 2007; 104(28): 11655 - 11659. [Abstract] [Full Text] [PDF]
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 Y. Robin-Lespinasse, S. Sentis, C. Kolytcheff, M.-C. Rostan, L. Corbo, and M. Le Romancer hCAF1, a new regulator of PRMT1-dependent arginine methylation J. Cell Sci., February 15, 2007; 120(4): 638 - 647. [Abstract] [Full Text] [PDF]
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D. Fu and K. Collins Human telomerase and Cajal body ribonucleoproteins share a unique specificity of Sm protein association Genes & Dev., March 1, 2006; 20(5): 531 - 536. [Abstract] [Full Text] [PDF]
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 D.J. BATTLE, M. KASIM, J. YONG, F. LOTTI, C.-K. LAU, J. MOUAIKEL, Z. ZHANG, K. HAN, L. WAN, and G. DREYFUSS The SMN Complex: An Assembly Machine for RNPs Cold Spring Harb Symp Quant Biol, January 1, 2006; 71(0): 313 - 320. [Abstract] [PDF]
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K. B. Shpargel and A. G. Matera Gemin proteins are required for efficient assembly of Sm-class ribonucleoproteins PNAS, November 29, 2005; 102(48): 17372 - 17377. [Abstract] [Full Text] [PDF]
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T. N. Azzouz, R. S. Pillai, C. Dapp, A. Chari, G. Meister, C. Kambach, U. Fischer, and D. Schumperli Toward an Assembly Line for U7 snRNPs: INTERACTIONS OF U7-SPECIFIC Lsm PROTEINS WITH PRMT5 AND SMN COMPLEXES J. Biol. Chem., October 14, 2005; 280(41): 34435 - 34440. [Abstract] [Full Text] [PDF]
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J. Cote and S. Richard Tudor Domains Bind Symmetrical Dimethylated Arginines J. Biol. Chem., August 5, 2005; 280(31): 28476 - 28483. [Abstract] [Full Text] [PDF]
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J. Herzog, Y. Maekawa, T. P. Cirrito, B. S. Illian, and E. R. Unanue Activated antigen-presenting cells select and present chemically modified peptides recognized by unique CD4 T cells PNAS, May 31, 2005; 102(22): 7928 - 7933. [Abstract] [Full Text] [PDF]
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 F.-M. Boisvert, C. A. Chenard, and S. Richard Protein Interfaces in Signaling Regulated by Arginine Methylation Sci. Signal., February 15, 2005; 2005(271): re2 - re2. [Abstract] [Full Text] [PDF]
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J.-H. Lee, J. R. Cook, Z.-H. Yang, O. Mirochnitchenko, S. I. Gunderson, A. M. Felix, N. Herth, R. Hoffmann, and S. Pestka PRMT7, a New Protein Arginine Methyltransferase That Synthesizes Symmetric Dimethylarginine J. Biol. Chem., February 4, 2005; 280(5): 3656 - 3664. [Abstract] [Full Text] [PDF]
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 M. Mahler, L. M. Stinton, and M. J. Fritzler Improved Serological Differentiation between Systemic Lupus Erythematosus and Mixed Connective Tissue Disease by Use of an SmD3 Peptide-Based Immunoassay Clin. Vaccine Immunol., January 1, 2005; 12(1): 107 - 113. [Abstract] [Full Text] [PDF]
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D. Cheng, N. Yadav, R. W. King, M. S. Swanson, E. J. Weinstein, and M. T. Bedford Small Molecule Regulators of Protein Arginine Methyltransferases J. Biol. Chem., June 4, 2004; 279(23): 23892 - 23899. [Abstract] [Full Text] [PDF]
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W. A. Smith, B. T. Schurter, F. Wong-Staal, and M. David Arginine Methylation of RNA Helicase A Determines Its Subcellular Localization J. Biol. Chem., May 28, 2004; 279(22): 22795 - 22798. [Abstract] [Full Text] [PDF]
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Q. Liu, X.-h. Liang, S. Uliel, M. Belahcen, R. Unger, and S. Michaeli Identification and Functional Characterization of Lsm Proteins in Trypanosoma brucei J. Biol. Chem., April 30, 2004; 279(18): 18210 - 18219. [Abstract] [Full Text] [PDF]
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M. Mandelboim, S. Barth, M. Biton, X.-h. Liang, and S. Michaeli Silencing of Sm Proteins in Trypanosoma brucei by RNA Interference Captured a Novel Cytoplasmic Intermediate in Spliced Leader RNA Biogenesis J. Biol. Chem., December 19, 2003; 278(51): 51469 - 51478. [Abstract] [Full Text] [PDF]
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T. N. AZZOUZ and D. SCHUMPERLI Evolutionary conservation of the U7 small nuclear ribonucleoprotein in Drosophila melanogaster RNA, December 1, 2003; 9(12): 1532 - 1541. [Abstract] [Full Text] [PDF]
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 F.-M. Boisvert, J. Cote, M.-C. Boulanger, and S. Richard A Proteomic Analysis of Arginine-methylated Protein Complexes Mol. Cell. Proteomics, December 1, 2003; 2(12): 1319 - 1330. [Abstract] [Full Text] [PDF]
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R. S. Pillai, M. Grimmler, G. Meister, C. L. Will, R. Luhrmann, U. Fischer, and D. Schumperli Unique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component, Lsm11, in histone RNA processing Genes & Dev., September 15, 2003; 17(18): 2321 - 2333. [Abstract] [Full Text] [PDF]
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C. XU, P. A. HENRY, A. SETYA, and M. F. HENRY In vivo analysis of nucleolar proteins modified by the yeast arginine methyltransferase Hmt1/Rmt1p RNA, June 1, 2003; 9(6): 746 - 759. [Abstract] [Full Text] [PDF]
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 P. S. C. Leung, C. Quan, O. Park, J. Van de Water, M. J. Kurth, M. H. Nantz, A. A. Ansari, R. L. Coppel, K. S. Lam, and M. E. Gershwin Immunization with a Xenobiotic 6-Bromohexanoate Bovine Serum Albumin Conjugate Induces Antimitochondrial Antibodies J. Immunol., May 15, 2003; 170(10): 5326 - 5332. [Abstract] [Full Text] [PDF]
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 W H Reeves, S Narain, and M Satoh Henry Kunkel, Stephanie Smith, clinical immunology, and split genes Lupus, March 1, 2003; 12(3): 213 - 217. [Abstract] [PDF]
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 J. Cote, F.-M. Boisvert, M.-C. Boulanger, M. T. Bedford, and S. Richard Sam68 RNA Binding Protein Is an In Vivo Substrate for Protein Arginine N-Methyltransferase 1 Mol. Biol. Cell, January 1, 2003; 14(1): 274 - 287. [Abstract] [Full Text]
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F.-M. Boisvert, J. Cote, M.-C. Boulanger, P. Cleroux, F. Bachand, C. Autexier, and S. Richard Symmetrical dimethylarginine methylation is required for the localization of SMN in Cajal bodies and pre-mRNA splicing J. Cell Biol., December 23, 2002; 159(6): 957 - 969. [Abstract] [Full Text] [PDF]
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S. E. Whitehead, K. W. Jones, X. Zhang, X. Cheng, R. M. Terns, and M. P. Terns Determinants of the Interaction of the Spinal Muscular Atrophy Disease Protein SMN with the Dimethylarginine-modified Box H/ACA Small Nucleolar Ribonucleoprotein GAR1 J. Biol. Chem., December 6, 2002; 277(50): 48087 - 48093. [Abstract] [Full Text] [PDF]
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J. Baccon, L. Pellizzoni, J. Rappsilber, M. Mann, and G. Dreyfuss Identification and Characterization of Gemin7, a Novel Component of the Survival of Motor Neuron Complex J. Biol. Chem., August 23, 2002; 277(35): 31957 - 31962. [Abstract] [Full Text] [PDF]
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U. Narayanan, J. K. Ospina, M. R. Frey, M. D. Hebert, and A. G. Matera SMN, the spinal muscular atrophy protein, forms a pre-import snRNP complex with snurportin1 and importin {beta} Hum. Mol. Genet., July 15, 2002; 11(15): 1785 - 1795. [Abstract] [Full Text] [PDF]
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W. J. Friesen, A. Wyce, S. Paushkin, L. Abel, J. Rappsilber, M. Mann, and G. Dreyfuss A Novel WD Repeat Protein Component of the Methylosome Binds Sm Proteins J. Biol. Chem., March 1, 2002; 277(10): 8243 - 8247. [Abstract] [Full Text] [PDF]
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M. T. McClain, P. A. Ramsland, K. M. Kaufman, and J. A. James Anti-Sm Autoantibodies in Systemic Lupus Target Highly Basic Surface Structures of Complexed Spliceosomal Autoantigens J. Immunol., February 15, 2002; 168(4): 2054 - 2062. [Abstract] [Full Text] [PDF]
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A. Frankel, N. Yadav, J. Lee, T. L. Branscombe, S. Clarke, and M. T. Bedford The Novel Human Protein Arginine N-Methyltransferase PRMT6 Is a Nuclear Enzyme Displaying Unique Substrate Specificity J. Biol. Chem., January 25, 2002; 277(5): 3537 - 3543. [Abstract] [Full Text] [PDF]
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W. J. Friesen, S. Paushkin, A. Wyce, S. Massenet, G. S. Pesiridis, G. Van Duyne, J. Rappsilber, M. Mann, and G. Dreyfuss The Methylosome, a 20S Complex Containing JBP1 and pICln, Produces Dimethylarginine-Modified Sm Proteins Mol. Cell. Biol., December 15, 2001; 21(24): 8289 - 8300. [Abstract] [Full Text] [PDF]
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M. D. Hebert, P. W. Szymczyk, K. B. Shpargel, and A. G. Matera Coilin forms the bridge between Cajal bodies and SMN, the Spinal Muscular Atrophy protein Genes & Dev., October 15, 2001; 15(20): 2720 - 2729. [Abstract] [Full Text] [PDF]
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K. W. Jones, K. Gorzynski, C. M. Hales, U. Fischer, F. Badbanchi, R. M. Terns, and M. P. Terns Direct Interaction of the Spinal Muscular Atrophy Disease Protein SMN with the Small Nucleolar RNA-associated Protein Fibrillarin J. Biol. Chem., October 12, 2001; 276(42): 38645 - 38651. [Abstract] [Full Text] [PDF]
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C. Mura, D. Cascio, M. R. Sawaya, and D. S. Eisenberg The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core PNAS, April 25, 2001; (2001) 91102298. [Abstract] [Full Text]
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Z. Palfi, S. Lücke, H.-W. Lahm, W. S. Lane, V. Kruft, E. Bragado-Nilsson, B. Séraphin, and A. Bindereif The spliceosomal snRNP core complex of Trypanosoma brucei: Cloning and functional analysis reveals seven Sm protein constituents PNAS, July 12, 2000; (2000) 150236097. [Abstract] [Full Text]
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A. Frankel and S. Clarke PRMT3 Is a Distinct Member of the Protein Arginine N-Methyltransferase Family. CONFERRAL OF SUBSTRATE SPECIFICITY BY A ZINC-FINGER DOMAIN J. Biol. Chem., October 13, 2000; 275(42): 32974 - 32982. [Abstract] [Full Text] [PDF]
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J. Rho, S. Choi, Y. R. Seong, W.-K. Cho, S. H. Kim, and D.-S. Im PRMT5, Which Forms Distinct Homo-oligomers, Is a Member of the Protein-arginine Methyltransferase Family J. Biol. Chem., March 30, 2001; 276(14): 11393 - 11401. [Abstract] [Full Text] [PDF]
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T. L. Branscombe, A. Frankel, J.-H. Lee, J. R. Cook, Z.-h. Yang, S. Pestka, and S. Clarke PRMT5 (Janus Kinase-binding Protein 1) Catalyzes the Formation of Symmetric Dimethylarginine Residues in Proteins J. Biol. Chem., August 24, 2001; 276(35): 32971 - 32976. [Abstract] [Full Text] [PDF]
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C. Mura, D. Cascio, M. R. Sawaya, and D. S. Eisenberg The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core PNAS, May 8, 2001; 98(10): 5532 - 5537. [Abstract] [Full Text] [PDF]
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Z. Palfi, S. Lucke, H.-W. Lahm, W. S. Lane, V. Kruft, E. Bragado-Nilsson, B. Seraphin, and A. Bindereif The spliceosomal snRNP core complex of Trypanosoma brucei: Cloning and functional analysis reveals seven Sm protein constituents PNAS, August 1, 2000; 97(16): 8967 - 8972. [Abstract] [Full Text] [PDF]
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