Professional Documents
Culture Documents
Karl Skriner,1 Wolfgang Hueber,2 Erhan Süleymanoglu,3 Elisabeth Höfler,2 Veit Krenn,4
Josef Smolen,5 and Günter Steiner6
Objective. To investigate which members of the 17% of patients with mixed connective tissue disease,
heterogeneous nuclear RNP (hnRNP) family are tar- and <10% of patients with other rheumatic disorders.
geted by autoantibodies from patients with systemic Epitope mapping studies showed the autoantibodies to
rheumatic diseases. be directed to conformational epitopes in the
Methods. Using a semipurified preparation of N-terminal RNA-binding part of AUF1. However, auto-
natural hnRNP proteins, 365 sera from patients with antibody binding did not interfere with RNA binding as
rheumatic diseases and control subjects were screened assessed by gel-shift assays. Immunohistochemical
by immunoblotting for the presence of autoantibodies. studies revealed AUF1 to be expressed in the cytoplasm
Bacterially expressed recombinant hnRNP D (AUF1) of RA synovial tissue as compared with nuclear staining
proteins were used for confirming the data obtained. in osteoarthritis and normal synovium, particularly in
Binding of RNA and autoantibody to AUF1 was inves- macrophages of the lining layer and in fibroblasts of the
tigated by gel retardation assays. Expression of AUF1 in sublining areas.
cultivated cells and synovial tissue was analyzed by
Conclusion. These data identify AUF1 proteins as
indirect immunofluorescence and immunohistochemis-
novel autoantigens in SLE and related autoimmune
try.
disorders. Because AUF1 proteins are major compo-
Results. Autoantibodies to AUF1 proteins were
nents of messenger RNA stability complexes, our find-
detected in 33% of patients with systemic lupus ery-
ings suggest that these complexes form a novel macro-
thematosus, 20% of patients with rheumatoid arthritis,
molecular target structure for autoantibodies in
Supported by the Center for Molecular Medicine of the
rheumatic autoimmune diseases.
Austrian Academy of Sciences, Vienna, the German Ministry of
Education and Research, the National Genome Research Network Autoantibodies to intracellular antigens are a
(grants NGFN-2.SIPAGE and NIE-S02T28), and the European Union
AutoCure (grant 018661). distinguishing feature of autoimmune rheumatic dis-
1
Karl Skriner, PhD: Charité University Medicine Berlin, eases such as systemic lupus erythematosus (SLE), pro-
Humboldt University and Free University, Berlin, Germany, and gressive systemic sclerosis (SSc; scleroderma), polymyo-
Medical University of Vienna, Vienna, Austria; 2Wolfgang Hueber,
MD (current address: Stanford University, Palo Alto, California), sitis, mixed connective tissue disease (MCTD), or
Elisabeth Höfler, MTA: Hietzing Hospital, Vienna, Austria; 3Erhan rheumatoid arthritis (RA) (1). These autoantibodies are
Süleymanoglu, PhD (current address: Gazi University, Ankara, Tur- typically directed to sets of proteins associated with
key): Institute of Medical Biochemistry and Medical University of
Vienna, Vienna, Austria; 4Veit Krenn, MD: Charité University Med- discrete supramolecular entities built up of either DNA
icine Berlin, Humboldt University and Free University, Berlin, Ger- or RNA and proteins, such as the nucleosome, the
many; 5Josef Smolen, MD: Medical University of Vienna, Hietzing
Hospital, and Ludwig Boltzmann Institute for Rheumatology and
centromere, the spliceosome, the ribosome, or other
Balneology, Vienna, Austria; 6Günter Steiner, PhD: Institute of Med- RNPs (2). Based on this observation, Tan and Hardin
ical Biochemistry, Medical University of Vienna, and Ludwig Boltz- (3,4) formulated the particle hypothesis of autoimmuni-
mann Institute for Rheumatology and Balneology, Vienna, Austria.
Address correspondence and reprint requests to Karl Skriner, zation, which postulates that the autoimmunogenic rep-
PhD, Charité University Medicine Berlin, Department of Rheumatol- ertoire of a given disease could be localized on a single
ogy and Clinical Immunology, Tucholskystrasse 2, 10117 Berlin, Ger- or a limited number of subcellular units, each of which is
many. E-mail: Karl.Skriner@charite.de.
Submitted for publication January 29, 2007; accepted in an assembly of multiple noncovalently linked molecules.
revised form October 5, 2007. In eukaryotic cells, protein-encoding genes are
511
512 SKRINER ET AL
transcribed by RNA polymerase II into large precursor (n ⫽ 10), and osteoarthritis (OA; n ⫽ 26), and healthy control
RNAs that are known as heterogeneous nuclear RNAs subjects (n ⫽ 25) were assessed. The sera were derived from
clinically and serologically well-characterized patients and
or pre–messenger RNA (mRNA). During their entire
were obtained from the serum bank of the 2nd Department of
nuclear life, these RNAs are associated with a set of Medicine, Hietzing Hospital, with institutional ethics approval.
proteins that have been termed heterogeneous nuclear All patients with RA fulfilled the 1987 revised criteria of the
RNPs (hnRNP), thereby forming large complexes whose American College of Rheumatology (ACR; formerly, the
composition may be specific for each mRNA (5). In American Rheumatism Association) (14), all patients with
SLE met the 1982 criteria of the ACR (15), and all patients
association with the small nuclear RNPs (snRNP) and
with MCTD met the criteria described by Alarcon-Segovia and
other protein factors, they form the spliceosome. Data Villareal (16).
obtained in recent years have revealed that apart from Preparation of natural hnRNP proteins. To obtain a
snRNP, several other splicesomal proteins are targeted semipurified preparation of hnRNP proteins, extracts were
by patients with systemic autoimmune diseases. Interest- prepared from HeLa nuclei and partially purified by heparin–
ingly, although snRNP and certain splicing factors such Sepharose chromatography, essentially as previously described
(17).
as serine/arginine-rich proteins appear to be targeted Oligonucleotides. Oligoribonucleotides and polymer-
mainly, if not exclusively, in patients with SLE or ase chain reaction (PCR) primers were obtained from the
MCTD, autoantibodies to hnRNP have also been ob- DNA synthesis and sequencing unit of the Vienna Biocenter.
served in patients with other rheumatic diseases, partic- The following PCR primers were used: for N10, GACGACG-
ularly RA (6,7). Thus, antibodies to hnRNP A2 (also ACAAGATGGCGGCGGCAGCGGCAACGGCG; for N20,
GACGACGACAAGATGGGCGGCTCGGCGGGCGAG-
known as the RA33 antigen), as well as antibodies to the CAG; for Ct256, GACGACGACAAGATGGCCATGTCGA-
closely related hnRNP A1, have been repeatedly found AGGAACAATAT; for Ct145, GAGGAGAAGCCCGGTT-
in patients with RA and those with SLE, and antibodies CAAAATAGCACAAAGCC; for N174, GAGGAGGAGA-
to hnRNP I have been described to occur especially in AGATGGCCATGAAAACAAAAGAGCCG; for Ct173,
patients with SSc (8,9). Apart from their established GAGGAGAAGCCCGGTTCATTTGGCCCTTTTAGG-
TCT; for N63, GACGACGACAAGATGTCGGAGGGGGA-
roles in pre-mRNA splicing, hnRNP are involved in CAAG-ATT; for Nt30, GACGACGACAAGATGGTGGCG-
cytoplasmic export of mature mRNA and in various GCGACACAGGG. For RNA binding studies, the oligoribo-
aspects of posttranscriptional regulation of gene expres- nucleotides (AUUUA)5 and AACUUGUGAUUAUUUAU-
sion, including translation and regulation of mRNA UAUUUAUUUAUUAUUUAUUUAUUUA (derived from
stability (10). the 3⬘-UTR of the TNF␣ mRNA) were used (18).
Cloning and expression of recombinant proteins. The
The hnRNP D proteins are a subgroup of at least complementary DNA (cDNA) encoding the entire sequence
4 proteins generated by alternative splicing, which are of AUF1 (a kind gift from Gary Brewer, School of Medicine,
also known as AUF1 (AU-rich element [ARE]–binding Wake Forest University) and a series of deletion mutants
factor 1) because they preferentially bind to adenine- constructed by cloning of PCR fragments were cloned into
and uridine-rich sequences in the 3⬘–untranslated region pET30 vectors (Novagen, Madison, WI) and expressed as
His-tagged fusion proteins, which were purified by Ni-chelate
(3⬘-UTR) of short-lived mRNA such as c-fos or tumor affinity chromatography (Clontech, Palo Alto, CA). Purity was
necrosis factor ␣ (TNF␣) mRNA (11). Binding of AUF1 ⬃95% as analyzed by sodium dodecyl sulfate–polyacrylamide
leads to destabilization of the mRNA, resulting in their gel electrophoresis (SDS-PAGE) and Coomassie protein stain-
rapid degradation by RNases (12). These proteins are ing. For RNA binding studies, fragments were cloned into the
closely related to the hnRNP A/B proteins, showing a pET-8c vector and expressed without a His tag. Non–His-
similar general structure, i.e., 2 adjacent RNA recogni- tagged proteins were purified by cation exchange chromatog-
raphy on CM Sepharose, as previously described (19). The
tion motives (RRMs), which are followed by a glycine- AUF1p45 cDNA was cloned into the pcDNA 3.1/NT-GFP-
rich C-terminal auxiliary domain (13). In this study, we TOPO vector (Invitrogen) and transfected into HeLa cells, as
present strong evidence for the existence of such auto- previously described (11).
antibodies to AUF1 and demonstrate these antibodies Gel electrophoresis and immunoblotting. Samples
to be directed to the functionally important N-terminal were separated on 12% SDS minigels and transferred to
region of the protein. nitrocellulose membranes (BAS53; Schleicher & Schuell, Das-
sel, Germany), as previously described (18). After blocking the
nitrocellulose with blocking buffer (3% nonfat dried milk in
PATIENTS AND METHODS PBS, pH 7.4) for 60 minutes, the blots were incubated for 30
minutes with sera diluted between 1:25 and 1:100 in the same
Patients. A total of 365 sera from patients with RA buffer. The blots were washed 3 times for 5 minutes with PBS
(n ⫽ 101), SLE (n ⫽ 70), MCTD (n ⫽ 30), SSc (n ⫽ 44), containing 0.1% Triton X-100 (PBS–Triton) and subsequently
polymyositis/dermatomyositis (n ⫽ 17), primary Sjögren’s syn- incubated for 30 minutes with an alkaline phosphatase–
drome (n ⫽ 11), reactive arthritis (n ⫽ 31), psoriatic arthritis coupled anti-human IgG (Fc) secondary antibody (Accurate,
AUF1 PROTEINS IN SLE AND RELATED AUTOIMMUNE DISORDERS 513
Westbury, NY), as previously described. All steps were per- graphed. For quantitative analysis, an electronic autoradio-
formed at room temperature. graphy system (InstantImager; Packard, Meriden, CT) was
Immunoaffinity purification of antibodies. Autoanti- used.
bodies to AUF1 from 1 patient with RA and 2 patients with Gel retardation assay. The binding and competition
SLE were affinity-purified by the blot elution technique, as assays were performed essentially as described above except
previously described (19). Autoantibodies to hnRNP A2 puri- that samples were not UV irradiated but were immediately
fied by the same method were used as controls. Briefly, transferred to 5% nondenaturing polyacrylamide gels run in
nitrocellulose strips containing the blotted antigens were incu- electrophoresis buffer (25 mM Tris HCl, 192 mM glycine, pH
bated overnight at 4°C with sera diluted 1:10 in blocking buffer, 8.3) and separated for 60 minutes at 10 V/cm. For supershift
washed thoroughly, cut into small pieces, and finally incubated experiments, the reactions were initially incubated for 15
for 2 minutes with 0.1M Tris, pH 10.5. The eluate was minutes at 30°C, subsequently 1 l of affinity-purified antibody
immediately neutralized with 1/10 volume of 1M Tris HCl, pH was added, and incubation continued for an additional 15
6.8. Eluted antibodies were concentrated in Centricon-30 tubes minutes. Finally, gels were dried and analyzed by autoradiog-
(Amicon, Danvers, MA) by centrifugation for 20 minutes at raphy.
5,000g. To estimate the yield, affinity-purified antibodies were Statistical analysis. Fisher’s exact test was used to
analyzed by SDS-PAGE and Coomassie blue staining, using analyze the significance of anti-AUF1 antibody prevalences
purified human IgG (Sigma, St. Louis, MO) as standard. between patient groups and between patients and healthy
Finally, eluted antibodies (100 ng/ml) were analyzed by immuno- control subjects and for analyzing correlations of anti-AUF1
blotting, using either nuclear extracts or purified AUF1 as autoantibodies with clinical features.
antigen.
Indirect immunofluorescence microscopy. Commer-
cial acetone-fixed HEp-2 cell slides (ANA HEp-2 slides; RESULTS
Generic Assays, Dahlewitz, Germany) were used in immuno-
fluorescence studies. Slides were incubated with serum (di- Autoantibodies to hnRNP D (AUF1) in sera from
luted 1:200 in PBS) or affinity-purified anti-AUF1 antibody patients with rheumatic diseases. In routine immuno-
(100 ng/ml in PBS) for 60 minutes at room temperature, blot analyses of patient sera, in which a preparation of
washed 3 times with PBS, and subsequently incubated for 30 semipurified hnRNP proteins was used as an antigenic
minutes with fluorescein isothiocyanate–conjugated goat anti- source, prominent autoreactivity to 2 proteins with
human IgG (Caltag, South San Francisco, CA).
Preparation of rabbit antibodies. AUF1-specific anti- estimated molecular masses of 45 kd and 42 kd, respec-
sera were generated by immunizing rabbits with a keyhole tively, was repeatedly observed, and was particularly
limpet hemocyanin–coupled AUF1-derived peptide (the pronounced in sera from patients with SLE. To further
N-terminal 12–amino acid motif contained in all 4 AUF1 investigate this finding, a total of 266 sera from patients
isoforms). Animals were immunized subcutaneously with 0.8 with various rheumatic diseases were selected from the
mg peptide and boosted intravenously with 0.6 mg peptide
every 6 weeks. Antisera were collected and were used either serum bank and analyzed by immunoblotting. In these
unpurified or peptide affinity-purified. Affinity-purified sera studies, ⬃30% of SLE sera and ⬃20% of RA sera were
were monospecific for AUF1, as tested by immunoblotting shown to be reactive with the 45/42 doublet (Figure 1A).
using HeLa cellular extracts. The molecular masses of the 2 proteins suggested that
Immunohistochemical analysis. For immunohisto- they belong to the subgroup of hnRNP D proteins,
chemical analyses, 1–3-m paraffin sections of synovial tissue
obtained from patients with RA, patients with OA, and normal which comprises 4 members with molecular masses
subjects were mounted on poly-L-lysin–coated slides (Oligene, between 38 kd and 45 kd generated by alternative
Berlin, Germany), incubated overnight at 58°C, and deparaf- splicing. These proteins are better known as AUF1,
finized with xylene. The affinity-purified anti-AUF1 rabbit because they selectively bind to certain AU-rich ele-
antibody was applied at a 1:1,500 dilution, and anti-AUF1 ments that are frequently present in the 3⬘-UTR of
binding was detected using the LSAB/AP kit (Dako, Glostrup,
Denmark). short-lived mRNA.
RNA protein crosslink assay. Equimolar amounts (0.3 Therefore, we expressed the 45-kd variant of
M) of 32P-labeled RNA oligonucleotide and purified recom- AUF1 in Escherichia coli and investigated by immuno-
binant AUF1p45 or deletion mutants, respectively, were mixed blotting whether sera reactive with the 45/42-kd antigen
and incubated at room temperature for 20 minutes in 20 l of also recognized the recombinant protein. In these stud-
binding buffer (10 mM Tris HCl, pH 7.5, 1 mM EDTA, 4%
glycerol, 0.1% Triton X-100, 10 mM 2-mercaptoethanol). The ies, the majority of positive sera (89%) were indeed
reaction mixture was transferred to a microtiter plate, put on reactive with recombinant AUF1. Moreover, antibodies
ice, and irradiated with an ultraviolet (UV) lamp (Stratalinker; affinity-purified from recombinant AUF1 cross-reacted
Stratagene, La Jolla, CA) at 254 nm at a dose of 9.9 mJ/mm2. with both the 45-kd and 42-kd antigens (Figure 1B), and
After the addition of Laemmli sample buffer (50 mM Tris HCl, antibodies affinity-purified from the natural proteins
pH 6.8, 2% weight/volume SDS, 10% volume/volume glycerol,
5% w/v 2-mercaptoethanol, 0.01% w/v bromophenol blue), the clearly recognized the recombinant protein (Figure 1C).
samples were boiled for 10 minutes and analyzed on 10% The 4 AUF1 isoforms differ by 2 insertions of 19
SDS–polyacrylamide gels. The gels were dried and autoradio- and 49 amino acids, respectively, which are contained in
514 SKRINER ET AL
Figure 1. Autoantibodies to AUF1 in sera from patients with systemic lupus erythematosus (SLE).
A, Immunoblot analysis of SLE sera using a semipurified preparation of heterogeneous nuclear
RNP proteins. Arrows indicate the positions of the 45-kd and 42-kd antigens corresponding to the
2 larger variants of AUF1. A human serum strongly reactive with both proteins was used as
reference (R) throughout the study. Pronounced anti-45/42 (AUF1) reactivities can be seen in lanes
2, 9, 13, 16, and 18. B, Immune reactivity of human autoantibodies with natural AUF1 purified from
recombinant protein p45 (lane 2). C, Recombinant 45-kd variant (AUF1p45) analyzed by Western
blotting. Serum antibodies were affinity-purified by blot elution from the native p45 (lane 2) and
p42 kd AUF1 (lane 3) proteins. Lane 1 (B and C) shows serum from a patient with rheumatoid
arthritis.
the first RRM and the C-terminal portion of the protein were not essential for antibody binding (results not
(11). In order to test whether these insertions were shown).
required for recognition by autoantibodies, the 3 shorter As visualized using indirect immunofluorescence
variants were analyzed by immunoblotting for reactivity microscopy, AUF1-positive sera produced a large speck-
with sera recognizing AUF1p45. All 4 proteins showed led nucleoplasmic staining sparing the nucleoli in inter-
comparable reactivity, demonstrating that the insertions phase cells (Figure 2A). To demonstrate the specificity
Table 1. Frequency of autoantibodies to recombinant AUF1, as Correlation of anti-AUF1 antibodies with other
determined by immunoblotting, in sera from patients and healthy autoantibodies. Sera from patients with RA or SLE are
controls*
known to contain antibodies to hnRNP A2 (9), which is
No. closely related to AUF1, showing a similar general
No. of (%)
structure and ⬃70% homology in the 2 RRMs. How-
Diagnosis sera positive
ever, although some sera contained autoantibodies to
Systemic lupus erythematosus 70 23 (33) both proteins, the correlation between the 2 antibody
Rheumatoid arthritis 101 20 (20)
Mixed connective tissue disease 30 5 (17) species did not reach the level of statistical significance,
Primary Sjögren’s syndrome 11 1 (9) in neither patients with RA nor patients with SLE. In
Polymyositis/dermatomyositis 17 0
Scleroderma† 44 0
patients with RA, there was also no correlation of
Psoriatic arthritis 10 0 anti-AUF1 with rheumatoid factor or antibodies to
Reactive arthritis 31 2 (7) citrullinated peptides. In patients with SLE, there was no
Osteoarthritis 26 1 (4)
Healthy controls 25 0 correlation with anti-DNA, anti-Ro, or anti-La autoan-
tibodies. Interestingly, however, an association was
* Specificity for systemic lupus erythematosus versus all other diseases,
90%; versus connective tissue diseases, 94%. found with anti-Sm antibodies, because among the 5
† Twenty-two patients had diffuse scleroderma, and 22 patients had anti-Sm–positive sera, 4 also contained antibodies to
limited scleroderma. AUF1 (P ⬍ 0.04). Significant associations of anti-AUF1
antibodies with clinical features were not observed in
patients with SLE, patients with RA, or patients with
MCTD.
of this pattern, antibodies affinity-purified from patient Epitope mapping. To identify the epitopes recog-
sera were used (Figure 2B). In addition to the nucleo- nized by anti-AUF1 autoantibodies, a series of deletion
plasmic staining, affinity-purified anti-AUF1 antibody mutants of AUF1p45 were generated. These fragments
stained discrete cytoplasmic foci, which were (by mor- were probed by immunoblotting with 21 selected sera
phologic criteria) clearly distinct from other organelles from patients with SLE (n ⫽ 7), patients with RA (n ⫽
such as mitochondria, endosomes/lysosomes, or the 9), and patients with MCTD (n ⫽ 5). Figure 3A shows a
Golgi complex. The location and size of discrete cyto- typical result obtained with serum from a patient with
SLE; this serum showed full reactivity with fragments
plasmic foci were similar to the size and location of
1–262, 10–262, and 1–173 and weak reactivity with
P-bodies (also known as GW bodies), and these foci
fragment 1–145, while no reactivity was observed with
concentrate enzymes involved in mRNA turnover and
any of the other fragments. Figure 3B shows the com-
sequester mRNA away from the translational machinery
bined results obtained with 11 fragments of AUF1. All
(20). To further analyze localization of AUF1, HeLa
sera showed comparable reactivity with the full-length
cells were transfected with green fluorescent protein
protein and deletion mutant 1–262, and the majority of
(GFP)–tagged AUF1p45, which showed a staining pat-
them (i.e., 16 of 21) were also fully reactive with
tern that was similar to that obtained with the affinity-
fragment 1–173 (containing the complete RRM 1),
purified anti-AUF1 antibody (Figure 2C).
suggesting that RRM 2 and the C-terminal part were not
To investigate the autoimmune response to essential for immunoreactivity.
AUF1 in greater detail, 365 sera from patients with In contrast, a short C-terminal truncation of
various rheumatic diseases were tested for reactivity RRM 1 (fragment 1–145) led to a significant reduction
with purified recombinant AUF1p45 (Table 1). These or complete loss of reactivity of all sera analyzed, and
studies largely confirmed the findings obtained with the fragment 1–70 (containing only the N-terminal domain)
natural proteins: IgG antibodies to AUF1 were observed was completely nonreactive. In a similar manner,
in one-third of sera from patients with SLE, in 20% of N-terminal truncations of fragment 1–262 gradually
sera from patients with RA, and in 17% of sera from reduced or abolished the reactivities. Thus, fragment
patients with MCTD, whereas no antibodies were found 10–262 was still recognized by 19 sera, and fragment
in sera from patients with scleroderma or polymyositis/ 30–262 was recognized by 14 of them, whereas fragment
dermatomyositis and, apart from a few exceptions (1 62–262 containing both RRMs but lacking the
patient with primary Sjögren’s syndrome, 2 patients with N-terminal domain was recognized by only 2 RA and 3
reactive arthritis, 1 patient with osteoarthritis), were MCTD sera. Only 2 of these sera recognized fragment
absent in patients with other rheumatic diseases. 105–262, while they showed no reactivity with fragment
516 SKRINER ET AL
Figure 3. Epitope mapping of AUF1. A, Reactivity of recombinant AUF1p45 fragments with a representative serum from a patient
with systemic lupus erythematosus (SLE). The positions of the NH2- and COOH-terminal amino acids of the fragments are indicated
at the top of the lanes. The top panel shows Coomassie blue staining, and the bottom panel shows results of immunoblot analysis. Lane
1, Full-length AUF1p45; lane 2, fragment 1–262 lacking the C-terminal auxiliary domain; lane 3, fragment 10–262; lane 4, fragment
20–262; lane 5, fragment 30–262; lane 6, fragment 62–262 lacking the N-terminal auxiliary domain; lane 7, fragment 1–173 comprising
the N-terminal domain and RNA recognition motif 1 (RRM 1); lane 8, fragment 1–145 lacking 28 C-terminal amino acids from RRM
1; lane 9, fragment 1–70 corresponding to the N-terminal auxiliary domain; lane 10, fragment 256–362 containing the glycine-rich
C-terminal auxiliary domain. B, Summary of the immunoblotting results obtained with 11 recombinant fragments of AUF1. The
N-terminal region of RRM 1 and RRM 2 and the glycine-rich auxiliary domain are schematically drawn; numbers above the AUF1
model designate the amino acid positions at the border of each domain. The amino acids bordering the deletion mutants are indicated.
The numbers of reactive sera are shown on the right side. The major epitope recognized by most sera was located between amino acids
1 and 173, and a minor epitope recognized by sera from some patients with rheumatoid arthritis (RA) or mixed connective tissue
disease (MCTD) was identified between amino acids 62 and 262. RBD ⫽ RNA binding domain.
137–262. No serum was reactive with either the and A1, because these proteins do not have similar
N-terminal or the C-terminal auxiliary domain alone. N-terminal domains (21).
Taken together, these analyses identified a major, Localization of the binding site for oligonucleo-
presumably discontinuous epitope composed of se- tides containing AREs. Rapid degradation of many
quences contained in the N-terminal auxiliary domain unstable mRNA is regulated in part by AREs, which are
and RRM 1 (Figure 3B). This unique epitope recogni- located in the 3⬘-UTR (22). It has been shown previously
tion explains the lack of cross-reactivity with hnRNP A2 that both cellular and recombinant AUF1 can bind
AUF1 PROTEINS IN SLE AND RELATED AUTOIMMUNE DISORDERS 517
Figure 4. Interaction of AUF1 with AU-rich element (ARE). A, Binding of AUF1 and
deletion mutants to the 32P-labeled oligoribonucleotide (AUUUA)4 representing a proto-
type ARE as determined by ultraviolet crosslinking assay. Crosslinked products were
resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and autoradio-
graphed. B, AUF1–ARE gel-shift assays performed with AUF1p45 and (AUUUA)4 or the
ARE regulatory sequence of human tumor necrosis factor ␣ (TNF␣) mRNA (TNF–ARE).
For supershift assays, affinity-purified patient antibodies to AUF1 or heterogeneous nuclear
RNP A2 (hnRNP A2; Ab2) were used. The anti-AUF1 antibody was able to shift both
AUF1–ARE complexes (lanes 2 and 7), while the affinity-purified anti–hnRNP A2 control
antibody did not (lane 5). Lane 1, Complex of AUF1 with TNF–ARE; lane 2, complex
formed by the addition of anti-AUF1 antibody; lane 3, competition with nonradioactive
TNF␣ oligoribonucleotide (10⫻ molar excess); lane 4, competition with 10⫻ molar excess
of -globin oligoribonucleotide; lane 5, addition of affinity-purified anti–hnRNP A2
autoantibody; lane 6, complex of AUF1 with (AUUUA)4; lane 7, complex formed by the
addition of anti-AUF1; lane 8, oligoribonucleotide (AUUUA)4. Cterm ⫽ C-terminal; COI
⫽ cold inhibition.
specifically to AREs. Using the oligonucleotide were in good agreement with previous findings from
(AUUUA)4 as a prototype ARE, the RNA-binding other investigators (21). Of note, fragment 1–173 (N-
properties of AUF1 fragments were investigated in UV terminal domain plus RRM 1), comprising the major
crosslinking assays (Figure 4A). Similar results were epitope, did not interact with the (AUUUA)4 oligo-
obtained with the p42 and p45 variants. Compared with nucleotide. Furthermore, anti-AUF1 antibodies from
the binding capacity of full-length proteins, the binding patients with SLE, RA, or MCTD did not inhibit RNA
capacity of fragment 1–262 was ⬃70%, while binding of binding (data not shown), indicating that the binding
the RNA to fragment 1–239 (lacking 17 amino acids sites for RNA and those for autoantibodies are different.
from the C-terminal part of RRM 2) was reduced to AUF1–ARE complexes are supershifted by anti-
⬃40%. Fragment 1–189 (lacking RRM 2) and fragment AUF1 autoantibodies. To investigate the simultaneous
137–262 (lacking most of RRM 1) showed only ⬃20% interaction of autoantibodies and ARE with AUF1,
binding capacity, and no binding was visible with frag- supershift experiments were performed in which the
ment 167–262 (corresponding to RRM 2) and fragment recombinant protein was incubated simultaneously with
1–173 (N-terminus plus RRM 1) (data not shown). In RNA oligonucleotides and sera or affinity-purified pa-
contrast, fragment 167–354, containing RRM 2 and the tient antibodies. In these experiments, both (AUUUA)4
entire C-terminal part, showed ⬃70% binding activity. and the ARE of human TNF␣ mRNA were used (Figure
The C-terminal auxiliary domain also contributes 4B); both sequences were supershifted by affinity-
to binding, because truncation of this part led to reduced purified anti-AUF1 antibodies but not by antibodies to
binding capacity, and the fragment containing RRM 2 hnRNP A2 obtained from the same patient. In control
plus the C-terminal domain showed a binding capacity competition experiments, unlabeled homologous ARE
similar to that of fragment 1–262. Thus, these results could abolish supercomplex formation, whereas a
518 SKRINER ET AL
ity of anti-AUF1–positive patients with SLE did not rather part of another structure formed during mRNA
have antibodies to Sm or U1 snRNP. Because in patients decay.
with SLE, anti-AUF1 antibodies occurred indepen- One of these structures might be the exosome.
dently of other serologic markers for this disorder such This is a large multiprotein complex of which proteins
as anti–double-stranded DNA or anti-Ro antibodies, such as AUF1, HuR, and TTP are part (28,29). These
they might have some diagnostic value for SLE, partic- proteins bind directly to the ARE before other proteins
ularly because they were absent or rare in other connec- associate. Binding of AUF1 seems to be essential to
tive tissue diseases such as scleroderma and form the exosome, and AUF1 is part of mRNA com-
polymyositis/dermatomyositis. Thus, their sensitivity and plexes that are degraded or exported. Interestingly, 2
specificity for SLE were 33% and 90%, respectively, human exosome components, PM–Scl-100 and PM–Scl-
which is comparable with anti–U1 snRNP antibodies. 75, are recognized by sera from patients with the
The detailed epitope mapping study performed polymyositis–scleroderma overlap syndrome (30,31).
with sera from patients with SLE, patients with RA, and However, the complete absence of anti-AUF1 antibod-
patients with MCTD revealed that the major antigenic ies in patients with scleroderma or polymyositis/
region is presumably conformation dependent. The ma- dermatomyositis along with the absence of antiexosomal
jor epitope appears to be composed of sequences con- antibodies in patients with SLE suggest that patients
tained in the unique N-terminal region, which is not with SLE do not target AUF1 contained in exosomal
shared by any other hnRNP protein, and part of RRM 1, complexes, while mRNA decay complexes do not appear
which shows ⬃70% homology with RRM 1 of hnRNP to form major target structures in scleroderma and
A2. This epitope was recognized by most sera, whereas a related disorders.
fragment containing RRM 2 was not reactive at all. This The investigation of AUF1 protein expression in
result explains the lack of cross-reactivity with the synovial tissue demonstrated that AUF1 proteins were
closely related hnRNP A2, whose major epitope is expressed in tissue from patients with RA, patients with
OA, and normal subjects. Of note, the number of cells
located in RRM 2 (19). No difference in epitope recog-
expressing the antigen was higher in RA synovial tissue
nition was observed between RA and SLE sera, while 3
than in OA and normal tissue samples. Interestingly, in
of 5 MCTD sera were reactive with a fragment contain-
RA but not OA synoviocytes, AUF1 proteins appeared
ing both RRMs, which was not recognized at all by SLE
to be expressed not only in the nucleus but also in the
sera and was recognized by only 2 of 9 sera from patients
cytoplasm, which might be related to their established
with RA. Remarkably, the C-terminal glycine-rich part
function in regulating mRNA decay of TNF␣ and other
of AUF1 was not recognized by autoantibodies, a find-
proinflammatory cytokines. Evidence from mice with
ing that is consistent with the results obtained previously altered cytokine mRNA stability, along with human
for hnRNP A2 (19). data, suggests that the imbalance between the stability
Mapping the binding site of the oligonucleotide and decay of inflammatory cytokine mRNA regulated by
containing the AUUUA decay sequence confirmed that AUF1 could represent a basic mechanism leading to
both RRMs and the unique N-terminal domain are autoimmunity (32,33).
required for interaction with this and other AREs. In conclusion, the AUF1 proteins have been
Because anti-AUF1 antibodies can bind simultaneously identified as a novel group of autoantigens in patients
with the RNA, as demonstrated by gel-shift assays, we with systemic autoimmune diseases, particularly SLE,
speculate that the autoimmune response might be di- RA, and MCTD. These findings suggest that anti-AUF1
rected to an initiating mRNA decay complex. Some of antibodies target the mRNA decay complex, which may
the enzymes involved in mRNA degradation are con- form another large RNP target structure in systemic
centrated in discrete cytoplasmic foci known as mRNA autoimmunity. We hypothesize that increased formation
processing bodies (P-bodies; also known as GW bodies) of such complexes (e.g., due to overexpression of insta-
(20,26,27). The most common clinical diagnosis of pa- ble mRNA such as those for interleukin-1 and TNF)
tients with anti-GW182 antibodies is Sjögren’s syn- may lead to pathologic autoimmune reactions against
drome, followed by neurologic disease and SLE. AUF1 AUF1 and other proteins of mRNA decay complexes.
might be part of this decay complex, because GFP-
tagged AUF1 and affinity-purified anti-AUF1 antibod- AUTHOR CONTRIBUTIONS
ies stained discrete cytoplasmic regions that might cor-
Dr. Skriner had full access to all of the data in the study and
respond to P-bodies. Thus, it remains to be shown takes responsibility for the integrity of the data and the accuracy of the
whether AUF1 is indeed localized in such bodies or is data analysis.
520 SKRINER ET AL