Group Title: Arthritis Research & Therapy
Title: Autoimmune targeting of key components of RNA interference
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Title: Autoimmune targeting of key components of RNA interference
Physical Description: Book
Language: English
Creator: Jakymiw, Andrew
Ikeda, Keigo
Fritzler, Marvin
Reeves, Westley
Satoh, Minoru
Chan, Edward
Publisher: Arthritis Research and Therapy
Publication Date: 2006
 Notes
Abstract: RNA interference (RNAi) is an evolutionarily conserved mechanism that is involved in the post-transcriptional silencing of genes. This process elicits the degradation or translational inhibition of mRNAs based on the complementarity with short interfering RNAs (siRNAs) or microRNAs (miRNAs). Recently, differential expression of specific miRNAs and disruption of the miRNA synthetic pathway have been implicated in cancer; however, their role in autoimmune disease remains largely unknown. Here, we report that anti-Su autoantibodies from human patients with rheumatic diseases and in a mouse model of autoimmunity recognize the human Argonaute (Ago) protein, hAgo2, the catalytic core enzyme in the RNAi pathway. More specifically, 91% (20/22) of the human anti-Su sera were shown to immunoprecipitate the full-length recombinant hAgo2 protein. Indirect immunofluorescence studies in HEp-2 cells demonstrated that anti-Su autoantibodies target cytoplasmic foci identified as GW bodies (GWBs) or mammalian P bodies, structures recently linked to RNAi function. Furthermore, anti-Su sera were also capable of immunoprecipitating additional key components of the RNAi pathway, including hAgo1, -3, -4, and Dicer. Together, these results demonstrate an autoimmune response to components of the RNAi pathway which could potentially implicate the involvement of an innate anti-viral response in the pathogenesis of autoantibody production.
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Research article

Autoimmune targeting of key components of RNA interference
Andrew Jakymiw1, Keigo Ikeda1, Marvin J Fritzler2, Westley H Reeves3, Minoru Satoh3 and
Edward KL Chan1


1Department of Oral Biology, University of Florida, 1600 S.W. Archer Road, Gainesville, FL, 32610, USA
2Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive N.W., Calgary, AB, T2N 4N1, Canada
3Division of Rheumatology and Clinical Immunology, Department of Medicine, and Department of Pathology, Immunology, and Laboratory Medicine,
University of Florida, 1600 S.W. Archer Road, Gainesville, FL, 32610, USA

Corresponding author: Edward KL Chan, echan@ufl.edu

Received: 7 Mar 2006 Revisions requested: 11 Apr 2006 Revisions received: 12 Apr 2006 Accepted: 19 Apr 2006 Published: 9 May 2006

Arthritis Research & Therapy 2006, 8:R87 (doi:1 0.11 86/arl 959)
This article is online at: http://arthritis-research.com/content/8/4/R87
C 2006 Jakymiw et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract


RNA interference (RNAi) is an evolutionarily conserved
mechanism that is involved in the post-transcriptional silencing
of genes. This process elicits the degradation or translational
inhibition of mRNAs based on the complementarity with short
interfering RNAs (siRNAs) or microRNAs (miRNAs). Recently,
differential expression of specific miRNAs and disruption of the
miRNA synthetic pathway have been implicated in cancer;
however, their role in autoimmune disease remains largely
unknown. Here, we report that anti-Su autoantibodies from
human patients with rheumatic diseases and in a mouse model
of autoimmunity recognize the human Argonaute (Ago) protein,
hAgo2, the catalytic core enzyme in the RNAi pathway. More


Introduction
The exact mechanisms and causes of autoimmune diseases
remain unknown. They are thought to develop when self-reac-
tive lymphocytes escape from tolerance and are activated or
when incomplete thymic and/or bone marrow clonal selection
or disruption of the energy of autoreactive lymphocytes per-
turb the delicate balance of non-self-antigen and self-antigen
recognition [1]. The disequilibrium between pro-inflammatory
and immunosuppressive cytokines is also thought to contrib-
ute to the autoimmune phenomenon [2].

Although our understanding of these specific disease proc-
esses is incomplete, human autoantibodies have proven very
useful for the discovery, identification, and elucidation of newly
described cellular components and macromolecules [3]. For
example, the identification and characterization of small


specifically, 910/0 (20/22) of the human anti-Su sera were shown
to immunoprecipitate the full-length recombinant hAgo2 protein.
Indirect immunofluorescence studies in HEp-2 cells
demonstrated that anti-Su autoantibodies target cytoplasmic
foci identified as GW bodies (GWBs) or mammalian P bodies,
structures recently linked to RNAi function. Furthermore, anti-Su
sera were also capable of immunoprecipitating additional key
components of the RNAi pathway, including hAgol, -3, -4, and
Dicer. Together, these results demonstrate an autoimmune
response to components of the RNAi pathway which could
potentially implicate the involvement of an innate anti-viral
response in the pathogenesis of autoantibody production.


nuclear ribonucleoproteins and the spliceosome were made
possible through the use of human autoantibodies [4].

Patients with systemic rheumatic diseases commonly produce
antibodies against specific classes of highly conserved RNA-
protein complexes. These include several known RNA-binding
autoantigens, such as SS-A/Ro, SS-B/La, Sm, and U1 RNP
[3]. RNA-binding proteins are of interest because they repre-
sent a class of novel regulators of gene expression. Their func-
tions include, but are not limited to, transcription, splicing,
translation, transport, stability, and degradation.

Recently, human autoantibodies were used to identify and
characterize a new protein named GW182 [5]. GW182 is an
mRNA-binding protein that is characterized by a highly repeti-
tive glycine (G) and tryptophan (W) domain at the amino ter-
minus. In addition, GW182 is associated with a subcellular


Page 1 of 8
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Ago = Argonaute; dsRNA = double-stranded RNA; EDTA = ethylenediaminetetraacetic acid; G = glycine; GWB = GW body; IIF = indirect immun-
ofluorescence; IP= immunoprecipitation; miRNA= microRNA; PCR = polymerase chain reaction; RISC = RNA-induced silencing complex; RNAi=
RNA interference; siRNA = short interfering RNA; SLE = systemic lupus deerythematosus; TnT = transcription and translation; W = tryptophan.







Arthritis Research & Therapy Vol 8 No 4 Jakymiw et al.



structure, the GW body (GWB) or mammalian P body, that is
involved in mRNA degradation [6,7]. More recently, knock-
down of GW182 and disruption of GWBs were demonstrated
to impair RNA interference (RNAi) or RNA silencing [8,9].

RNAi is an evolutionarily conserved mechanism involved in the
post-transcriptional regulation of gene expression in many
eukaryotes [10]. It was initially recognized as an anti-viral
mechanism that protected organisms from RNA viruses [11]
or the random integration of transposable elements [10]. How-
ever, not until the discovery that plants and animals encode
small RNA molecules referred to as microRNAs (miRNAs) did
it become apparent that this mechanism was also responsible
for the post-transcriptional regulation of gene expression
[10,12].

RNAi is triggered by double-stranded RNA (dsRNA) precur-
sors that are rapidly processed into small RNA duplexes of
approximately 21 nucleotides in length by a dsRNA-specific
endonuclease termed Dicer [10]. These small RNA duplexes
commonly referred to as short interfering RNAs (siRNAs) or
miRNAs incorporate into the RNA-induced silencing complex
(RISC). Upon binding to RISC, one of the RNA strands then
disassociates and subsequently activates the complex. The
single-strand siRNA/miRNA within RISC then guides and ulti-
mately cleaves or represses the translation of target mRNAs
[10].

Some of the proteins most consistently found in RISC are the
highly conserved Argonaute (Ago) proteins [12]. There are
eight proteins in the human Ago family [13], four of which,
hAgol-4, have been demonstrated to associate with siRNAs/
miRNAs in humans [14]. However, only hAgo2 has been dem-
onstrated to possess the catalytic cleavage activity associated
with RNAi [15,16]. Interestingly, hAgo2 has been recently
demonstrated to associate with GW182 and localize to
GWBs [8,9,14,17].

To date, the most commonly identified diagnoses of patients
with autoantibodies to GW182 and GWBs are Sjdgren's syn-
drome, mixed motor/sensory neuropathy, and systemic lupus
erythematosus (SLE) [18]. However, autoantibodies to GWBs
with other antigen specificities have also recently been identi-
fied in patient sera [19-22], in particular from a subset of
patients with primary biliary cirrhosis [19]. Therefore, the iden-
tification of autoantibodies targeting GW182 and GWBs
[5,18] and their recent links with RNAi [8,9,14,17] suggest
that other components of the RNAi pathway may potentially be
targets of autoimmunity. In this report, we show that the previ-
ously reported anti-Su autoantibody [23,24] targets hAgo2
and other key components of the RNAi machinery. Further-
more, we demonstrate that anti-Su autoantibodies stain
GWBs in human cells. The significance of this study is that it
identifies autoimmune responses to components of the RNAi


machinery and provides insights into systemic rheumatic dis-
eases associated with the Su antigen.

Materials and methods
Antibodies and sera
Human sera used in this study were obtained from serum
banks at the Advanced Diagnostics Laboratory, University of
Calgary (Calgary, AB, Canada), the University of Florida
Center for Autoimmune Diseases (Gainesville, FL, USA), and
the University of North Carolina Hospitals (Chapel Hill, NC,
USA). Murine sera were obtained from BALB/c mice prior to
or after pristane injection [25]. Murine monoclonal antibodies
to GW182 (4B6) were from CytoStore Inc. (Calgary, AB, Can-
ada). Human and murine anti-Su autoimmune sera were iden-
tified based on specific reactivity to the 100/102- and 200-
kDa Su antigens by immunoprecipitation (IP) of radiolabeled
K562 (human erythroleukemia) cell extracts, SDS-PAGE, and
autoradiography [23]. Prototype human anti-GW182 sera
were described previously [5,8,18]. These studies were
approved by the institutional review boards and institutional
animal care and use committees of the University of Florida,
the University of North Carolina, and the University of Calgary.

Plasmid DNA constructs
The hAgo2 cDNA in pCMV-SPORT vector was obtained from
Dr. Tom Hobman (University of Alberta, Edmonton, AB, Can-
ada) [26]. The hAgol (clone 30344513; GenBank
BC063275) and hAgo4 (clone 4373725; GenBank
BF979523) cDNAs in pBluescript were purchased from
Open Biosystems (Huntsville, AL, USA). For both hAgol and
hAgo4, GC-rich regions in the 5'-untranslated region were
deleted to enhance the in vitro transcription and translation
(TnT) reaction. Briefly, the hAgol plasmid was digested with
EcoRI and Smal, purified, filled in with Klenow polymerase,
and religated. Similarly, the hAgo4 plasmid was digested with
Notl and Ncol, purified, and ligated with a linker containing an
EcoRV cut site: 5'-GGCCGATATCGTGC-3' and 5'-CAT-
GGCACGATATC-3'. The hAgo3 cDNA (clone
CSODB008YP10; GenBank AL522515) in the pCMV-
SPORT6 vector was purchased from Invitrogen (Carlsbad,
CA, USA). The Dicer insert (KIAA0928; GenBank X52328) in
pBluescript was obtained from the Kazusa DNA Research
Institute (Chiba, Japan). The Dicer coding region was recloned
by polymerase chain reaction (PCR) amplification using the
following two PCR primers: 5'-CGATACAGTCGACGCCAC-
CATGGAAAGCCCTGCTTTGCAACC-3' and 5'-
CCAATACGGCACGACAGTC-3'. All constructs were con-
firmed by DNA sequencing performed by the University of Flor-
ida Interdisciplinary Center for Biotechnology Research core
laboratory.

Fluorescence microscopy
Indirect immunofluorescence (IIF) analysis was previously
described [8]. Briefly, the primary antibodies to the following
proteins were used: GW182 (human serum, 1:200-1:6,000;


Page 2 of 8
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Figure 1


anti-GWB


C,%J 04C\I N C\1J 04
MI M M
12345
1in 2


GW182v 0UUI* *Il1 u t
116-
97- ap muimm-o ~-1 OOkD doublet
66-

Immunoprecipitation analysis of human GW body (GWB)-positive
autoimmune sera. Analysis of human GWB-positive sera demonstrated
immunoprecipitation of GW182 from [35S]-methionine-labeled HeLa
cell extracts (lanes 1 -4), except for serum (H26) (lane 5). An approxi-
mately 100-kDa protein doublet (lanes 4 and 5) and a weak approxi-
mately 200-kDa protein (lane 4) were observed in some of these
immunoprecipitations. These immunoprecipitated proteins resembled
the immunoprecipitation characteristics of the Su autoantigen. Note
that a control normal human serum did not immunoprecipitate any of
the proteins described above (not shown).

murine monoclonal 4B6 (neat)); Su (human sera, 1:100-
1:500; mouse sera, 1:100-1:500). The secondary antibodies
were anti-mouse or human immunoglobulin G fluorochrome-
conjugated goat antibodies, Alexa Fluor 488 (1:400) and
Alexa Fluor 568 (1:400) (Invitrogen). Anti-Su sera were ana-
lyzed initially at 1:100, 1,200, 1:500, and 1:1,000 using HEp-
2 cells (Immuno Concepts N.A. Ltd., Sacramento, CA, USA).

In vitro TnT and IP
The protocol for in vitro TnT and IP was previously described
[18]. TnT reactions were performed in the presence of [35S]-
methionine. The IPs of hAgol-4 and Dicer TnT products were
performed using 0.5 M NaCI NET/NP40 buffer (50 mM Tris-
HCI [pH 7.5], 500 mM NaCI, 2 mM ethylenediaminetetraace-
tic acid (EDTA), 0.3% Nonidet P-40) and NET2+F buffer (50
mM Tris-HCI [pH 7.4], 150 mM NaCI, 5 mM EDTA, 0.5% Non-
idet P-40, 0.5% deoxycholic acid, 0.1% SDS, 0.02% sodium
azide) containing Complete protease cocktail inhibitors
(Roche, Basel, Switzerland), respectively. Cell labeling and IP
of proteins from [35S]-methionine-labeled HeLa or K562 cell
extracts using human and/or murine sera were described pre-
viously [5,23].

Results and Discussion
The Su antigen localizes to cytoplasmic structures
identified as GWBs
During our exploration of the biology of GWBs and RNAi,
hAgo2, an approximately 100-kDa protein, was demonstrated
to coimmunoprecipitate with GW182 and localize to GWBs
[8]. Studies using radiolabeled human cell extracts confirmed
the presence of an approximately 100-kDa protein that was


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Available online http://arthritis-research.com/content/8/4/R87



immunoprecipitated by some human GWB-positive autoim-
mune sera (Figure 1). Interestingly, we noted that the immuno-
precipitated protein exhibited IP characteristics similar to the
elusive Su autoantigen, which is part of a macromolecular
complex that consists of a 100/102-kDa protein doublet [23].
Based on these findings, we hypothesized that hAgo2 could
be the approximately 100-kDa Su autoantigen. If this hypothe-
sis were proven to be true, it would link a known cellular com-
ponent of the RNAi machinery to autoimmunity and the
induction of specific autoantibodies directed toward the Su
autoantigen.

Autoantibodies to the Su antigen were initially identified and
characterized in 1984 and reported to be specifically associ-
ated with SLE in both humans and mice [24,27]. However, a
more extensive study later demonstrated that the Su antigen
was a frequent target of autoantibodies in a variety of systemic
rheumatic diseases [23].

Several monospecific anti-Su human and mouse sera have
been demonstrated to exhibit a negative fluorescent anti-
nuclear antibody staining, although in a number of cases the
sera have been shown to stain the cytoplasm of cells [23].
Knowing that both endogenous and exogenously introduced
forms of hAgo2 localized to GWBs [8,9,14,17], we hypothe-
sized that the approximately 100-kDa Su antigen, if it were
hAgo2, would also localize to small discrete structures within
the cytoplasm of the cells, which are characteristic of GWBs.
Initial analysis of a human anti-Su autoimmune serum in HEp-
2 cells by IIF demonstrated that the serum stained discrete
cytoplasmic foci; these foci were determined to be GWBs by
containing with a GWB marker (Figure 2, top). Because pris-
tane has previously been demonstrated to induce the produc-
tion of anti-Su antibodies in approximately 50% of BALB/c
mice [25], we used an anti-Su serum from a pristane-induced
autoimmune mouse as further evidence in support of the
potential specificity of anti-Su serum for GWBs. Testing of this
serum by IIF independently suggested that the Su antigen is
enriched in GWBs (Figure 2, bottom). The failure of a previous
report [23] to observe GWB staining was most likely related
to the fixation protocol required to preserve GWBs within the
cytoplasm. The anti-GWB IIF titers for anti-Su sera ranged
from 1:200 to 1:500. In addition to the GWB localization, dif-
fuse cytoplasmic staining was observed for both human and
mouse anti-Su sera. This result is not surprising; similar cellular
staining patterns have been observed for hAgo2/RISC
[14,17], and it has been proposed that hAgo2/RISC shuttles
between the cytoplasm and cytoplasmic bodies [17].
Together, these data supported the notion that anti-Su sera
were capable of recognizing an antigen that localized to
GWBs, a known characteristic of hAgo2.

The Su antigen is biochemically similar to hAgo2
In previous biochemical studies, the Su antigen was demon-
strated to be a macromolecular complex consisting of 100/








Arthritis Research & Therapy Vol 8 No 4 Jakymiw et al.


Figure 2


a-Su


H100^^^H^r


a-GW182


Merge


Human and mouse anti-Su sera stain cytoplasmic foci identified as GW bodies (arrows). (top) HEp-2 cells were stained with human anti-Su serum
(H1 00) and anti-GW1 82 monoclonal antibody (4B6). Bar, 10 um. (bottom) Hep-2 cells were stained with mouse anti-Su (931 0B) and human anti-
GW182 (H20) sera. Note that the prototype human anti-GW1 82 (H20) serum is known to have additional nuclear-envelope antibodies [35]. Bar, 20
uim.

Table 1


Serological features of human and mouse anti-Su sera


K562 cell extract IP


GWB foci 100/102-kDa Su


Human anti-Su (n= 22)
Mouse anti-Su (n= 7)
Human anti-GW182 (n = 2)
Human autoimmune non-anti-Su (n = 10)
Normal human (n = 10)
Pre-immune mouse (n = 6)


91%/
1 00%
1 00%


100%
100%
50%
0%
0%
0%


TnT IP


200-kDa Su

82%/
100%/
50%/
0%/
0%/
0%/


hAgo2

91%
100%a/
50%
0%
0%
0%0


Dicer

36%


100%
0%
0%,
0%d


an = 5, bn = 2, on = 4, dn = 1. GWB, GW body; IIF, indirect immunofluorescence of HEp-2 cells; IP, immunoprecipitation; TnT, in vitro
transcription and translation.


102- and 200-kDa proteins [23], the former being consistent
with the approximately 100-kDa molecular mass of hAgo2. To
confirm that the Su antigen had a molecular mass similar to
that of hAgo2, a human prototype anti-Su serum was used to
immunoprecipitate the 100-kDa protein from a human (K562)
cell extract, after which the immunoprecipitated product was
compared with that of an in vitro translated hAgo2. SDS-
PAGE analysis clearly demonstrated that the Su antigen
immunoprecipitated by the human anti-Su serum comigrated
with the in vitro translated hAgo2 (Figure 3a). Furthermore,
human anti-Su sera and pristane-induced autoimmune anti-Su
mouse sera immunoprecipitated the in vitro translated hAgo2,
whereas human and mouse control sera did not (Figure 3b).


For this experiment and subsequent IP experiments involving
in vitro translated products, in vitro translated Luciferase pro-
tein was added to the IP mix to demonstrate specificity of anti-
Su interactions. The inability of the anti-Su sera to immunopre-
cipitate the in vitro translated Luciferase product in the exper-
iment described above demonstrated the specificity of the
anti-Su sera for hAgo2. Taken together, these data strongly
supported the hypothesis that the approximately 100-kDa Su
antigen was hAgo2.

Human and mouse anti-Su autoimmune sera recognize other
members of the Ago protein family. Because the Su antigen
has been characterized as a macromolecular complex that


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Available online http://arthritis-research.com/content/8/4/R87


consists of a doublet of approximately 100- and 102-kDa pro-
teins [23], it is possible that the 102-kDa protein is a post-
translationally modified form of the 100-kDa protein. It has also
been suggested that the two components most likely repre-
sent distinct polypeptides, because a few anti-Su sera have
been identified that recognize the 100-kDa, but not the 102-
kDa, component [23]. This observation prompted us to test
whether other members of the Ago protein family were tar-
geted by anti-Su sera. IP of full length in vitro translated
hAgol, -3, and -4 using either human anti-Su autoimmune
serum or pristane-induced autoimmune mouse serum demon-
strated that either sera was capable of immunoprecipitating
these additional Ago family members in addition to hAgo2
(Figure 4). The inability of the anti-Su sera to immunoprecipi-
tate the in vitro translated Luciferase product once again dem-
onstrated the specificity of the binding of anti-Su sera for the
family of Ago proteins in the IP reaction. Analysis of five addi-
tional human anti-Su (hAgo2-positive) sera similarly demon-
strated that each serum was capable of recognizing the other
three Ago family members. These results were not surprising
and are consistent with the concept of inter-molecular epitope
spreading [28], because Ago family members are known to
reside and associate together in a macromolecular RNA
silencing complex [14,17]. It is also possible that the sera
react with a common epitope in the Ago proteins by virtue of
the high degree of sequence homology (approximately 80%
identity) exhibited between the Ago proteins [13]. Regardless
of the type of interaction that occurs between the autoimmune
sera and these additional Ago family members, the important
point to note is that the anti-Su autoimmune sera target key
components of the RNAi machinery.

Human anti-Su autoimmune sera recognize Dicer
Besides the core Ago proteins and the small RNA compo-
nents of RISC, other proteins have been identified in higher
molecular mass forms of the purified complex [10]. These
larger forms are thought to be due to the weak and/or transient
association of proteins involved in the initial processing of
dsRNA (for example, Dicer) [10]. The sedimentation behavior
of the Su antigen suggests that it exists as a large protein com-
plex that carries 100/102- and 200-kDa proteins [23]. With
the identification of the approximately 100-kDa protein as
hAgo2, the obvious candidate for the 200-kDa protein was
Dicer. Dicer, an approximately 210-kDa protein, has been pre-
viously demonstrated to associate with hAgo2 [26]. To test
whether the 200-kDa component of the Su antigen had a
molecular mass similar to that of Dicer, a human anti-Su serum
was used to immunoprecipitate the 200-kDa protein from a
human (K562) cell extract, after which the immunoprecipitated
product was compared with that of an in vitro translated Dicer.
SDS-PAGE analysis demonstrated that the 200-kDa Su anti-
gen immunoprecipitated by the human anti-Su serum comi-
grated with the in vitro translated Dicer (Figure 5a).
Furthermore, the specificity of the reaction was demonstrated
when the human anti-Su sera immunoprecipitated the in vitro


Figure 3


(a)

0

a
1 2
205-



116-


"*h


hAgo2


Luciferase
J) 0
)3 -. -"
Su200kD _I 0400
+ ooCC S
C 4

< II I-
Su 102kD Z s t G 5
SSu 10kD
go2Su1kD 1 2 3 4 5 6 7
Ago2


-116
gR --97


hAgo2 -

Luciferase -p


Human and mouse anti-Su sera recognize in vitro translated hAgo2
protein. (a) K562 cells were radiolabeled and extract was immunopre-
cipitated with human anti-Su serum (lane 2) and compared with the
migration pattern of the in vitro synthesized hAgo2 product (lane 1). (b)
Immunoprecipitation of in vitro translated hAgo2 product using human
anti-GW182 serum (lane 3), human anti-Su sera (lanes 4 and 5), and
mouse anti-Su serum (lane 7) compared with normal human serum
(NHS) and pre-immune mouse serum (PIM) (lanes 2 and 6, respec-
tively). Lane 1 represents the in vitro transcription and translation mix
prior to immunoprecipitation.


translated Dicer, but the human control serum did not (Figure
5b). The inability of the anti-Su sera to immunoprecipitate the
in vitro translated Luciferase product also demonstrated the
specificity of the anti-Su sera for Dicer. Cumulatively, these
data strongly suggested Dicer as an additional molecular tar-
get of anti-Su sera; however, further studies, such as analysis
of the 200-kDa protein by IP-mass spectroscopy, will be
needed to determine whether Dicer is the true 200-kDa Su
antigen.

Serological characteristics of anti-Su autoimmune sera
To ascertain a broader perspective of the serological proper-
ties of anti-Su sera, 22 human and seven pristane-induced
mouse anti-Su sera were further characterized and compared
with other human Su-negative autoimmune (n = 10) or normal
human (n = 10) and mouse (n = 6) sera (Table 1). Autoim-
mune anti-Su sera were confirmed by IP of the characteristic
macromolecular complex, which consists of the doublet of
approximately 100/102- and 200-kDa proteins. IIF staining in
HEp-2 cells demonstrated that of the 22 human anti-Su sera,
20 (91%) stained cytoplasmic foci that were characteristic of
GWBs. Random selection of seven of these positive sera
demonstrated that all seven (100%) showed colocalization
with monoclonal antibodies to GW182. Of the human anti-Su


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Arthritis Research & Therapy Vol 8 No 4 Jakymiw et al.


Figure 4


Control Human Normal Mouse Pre-immune


(Ago +


(a-Su


Luciferase) (H21) Serum


Human a-Su Mouse


Serum


Ago


116 -
hAgol.
97 -

66 -
Luciferase "

31 -


1 2 3 4 1 23 4 1 2 3 4 1 2 3 4 1 23 4


Human and mouse anti-Su sera immunoprecipitate all four Argonaute (Ago) family members. In vitro translated hAgol, -2, -3, and -4 were immuno-
precipitated using both human and mouse anti-Su sera in comparison with both normal human and pre-immune mouse sera. As a control, the hAgo
in vitro transcription and translation mixes were also loaded for migration comparisons.


Figure 5


a) (D

S-Su
S,- Dicer + Luciferase






*1- Su200k Dicer -205
116- ASu 102kD
9 'OSu 10CkD -97
9 -i~~-116
--97
66-- Lucilerase
SLucfera .-, -66





Human anti-Su sera immunoprecipitate in vitro translated Dicer protein.
(a) K562 cells were radiolabeled and extract was immunoprecipitated
with human anti-Su serum (lane 2) and compared with the migration
pattern of the in vitro synthesized Dicer product (lane 1). (b) Immuno-
precipitation of in vitro translated Dicer product using human anti-Su
sera (lanes 3-6) compared with normal human serum (NHS) (lane 2).
Lane 1 represents the in vitro transcription and translation mix prior to
immunoprecipitation.

sera, 91% (20/22) and 36% (8/22) recognized in vitro trans-
lated hAgo2 and Dicer, respectively. Similarly, 100% (7/7) of
the seven pristane-induced autoimmune mouse sera also
stained GWBs, and of the tested sera, 100% (5/5) were
hAgo2-positive; however, none (0/2) were Dicer-positive.
None (0/16) of the normal human and pre-immune mouse sera


stained cytoplasmic structures characteristic of GWBs, and
none (0/10) of the Su-negative autoimmune sera stained
GWB structures. Of the tested sera, none were reactive with
in vitro translated hAgo2 (0/24) or Dicer (0/9). The signifi-
cance of these results is that anti-Su autoimmune sera appear
to be uniquely defined by their ability to stain GWBs within the
cytoplasm of cells and recognize the hAgo2 protein, thus mak-
ing them readily identifiable. During the course of our serolog-
ical studies, we also determined that one of the two prototype
human anti-GW182 sera was also positive for anti-Su antibod-
ies, further demonstrating the close association and potential
overlap between these two sets of autoimmune sera.

In summary, these data identify the Su autoantigen as a mac-
romolecular complex closely associated with the GWB struc-
ture and the RNAi pathway. Furthermore, these data
demonstrate that anti-Su autoantibodies target key compo-
nents of the RNA silencing machinery, in particular hAgo2.
However, further studies will be required to better ascertain
the extent of anti-Su autoantibody reactivity with other mem-
bers of the Ago family and definitively determine whether Dicer
is unambiguously the 200-kDa protein commonly associated
with the Su antigen. Regardless, the association between
RNAi and systemic autoimmune diseases is potentially signifi-
cant due to the growing connection between RNAi and
viruses. RNAi is an innate anti-viral response found in certain
plant and invertebrate species and remains an evolutionarily
conserved mechanism in many eukaryotes. Its role as a natural
anti-viral response in mammals has been postulated and is
supported by the evidence that mammalian viruses encode
suppressors of RNAi [29,30]. Even though at present the clin-
ical significance of anti-Su autoantibodies is not apparent, it is
intriguing to speculate that viruses may promote the develop-
ment of autoimmunity via their association with components of


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the RNAi pathway. Given that RNAi is linked to the interferon
system [31], viral suppressors inhibit RNAi [29,30], viruses
encode miRNAs [32], and virus-like particles associate with
GWB/P-body components [33], it is not surprising that
GWBs and thus RNAi protein and/or nucleic acid compo-
nents develop into targets of autoimmunity.

Conclusion
Our work links key components of the RNAi machinery with
autoimmune disease. Moreover, our study identifies a murine
model of autoimmunity that has the potential to advance our
understanding of autoimmune responses to components of
the RNAi pathway. Interestingly, pristane treatment of mice
has been demonstrated to activate endogenous retroviruses
[34]. Therefore, future studies with a focus on the interplay
among RNAi, viruses, and autoimmunity should help clarify
whether RNAi itself or its association with an invading virus is
directly linked to the pathogenesis of systemic autoimmune
diseases.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
AJ carried out the initial IIF and TnT-IP studies, participated in
the serological testing and in the design of the study, and
drafted the manuscript. KI carried out TnT-IP studies, com-
pleted the serological testing, and participated in the design of
the study. MS carried out the IP studies and participated in the
design of the study. MJF, WHR, and MS collected and classi-
fied the sera and also helped edit the manuscript. EKLC con-
ceived of the study, participated in its design and coordination,
and edited the manuscript. All authors read and approved the
final manuscript.

Acknowledgements
This work was supported in part with resources and the use of facilities
at the Malcom Randall VA Medical Center, Gainesville, FL, National Insti-
tutes of Health grants A139645, A144074, A147859, AR07603,
AR40391, AR42455, AR44731, AR50661, AR51766,, and
M01 R00082, State of Florida funds to the Center for Autoimmune Dis-
eases, and the Canadian Institutes for Health Research grant MOP-
38034. MJF holds the Arthritis Society Chair at the University of Calgary.

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