Autoimmune targeting of key components of RNA interference
© Jakymiw et al.; licensee BioMed Central Ltd. 2006
Received: 7 March 2006
Accepted: 19 April 2006
Published: 9 May 2006
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.
The exact mechanisms and causes of autoimmune diseases remain unknown. They are thought to develop when self-reactive lymphocytes escape from tolerance and are activated or when incomplete thymic and/or bone marrow clonal selection or disruption of the anergy of autoreactive lymphocytes perturb the delicate balance of non-self-antigen and self-antigen recognition . The disequilibrium between pro-inflammatory and immunosuppressive cytokines is also thought to contribute to the autoimmune phenomenon .
Although our understanding of these specific disease processes is incomplete, human autoantibodies have proven very useful for the discovery, identification, and elucidation of newly described cellular components and macromolecules . For example, the identification and characterization of small nuclear ribonucleoproteins and the spliceosome were made possible through the use of human autoantibodies .
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 . RNA-binding proteins are of interest because they represent a class of novel regulators of gene expression. Their functions 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 . GW182 is an mRNA-binding protein that is characterized by a highly repetitive glycine (G) and tryptophan (W) domain at the amino terminus. In addition, GW182 is associated with a subcellular structure, the GW body (GWB) or mammalian P body, that is involved in mRNA degradation [6, 7]. More recently, knockdown 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 . It was initially recognized as an anti-viral mechanism that protected organisms from RNA viruses  or the random integration of transposable elements . However, 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) precursors that are rapidly processed into small RNA duplexes of approximately 21 nucleotides in length by a dsRNA-specific endonuclease termed Dicer . 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 ultimately cleaves or represses the translation of target mRNAs .
Some of the proteins most consistently found in RISC are the highly conserved Argonaute (Ago) proteins . There are eight proteins in the human Ago family , four of which, hAgo1-4, have been demonstrated to associate with siRNAs/miRNAs in humans . However, only hAgo2 has been demonstrated 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 Sjögren's syndrome, mixed motor/sensory neuropathy, and systemic lupus erythematosus (SLE) . However, autoantibodies to GWBs with other antigen specificities have also recently been identified in patient sera [19–22], in particular from a subset of patients with primary biliary cirrhosis . Therefore, the identification 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 previously reported anti-Su autoantibody [23, 24] targets hAgo2 and other key components of the RNAi machinery. Furthermore, 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 diseases 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 . Murine monoclonal antibodies to GW182 (4B6) were from CytoStore Inc. (Calgary, AB, Canada). Human and murine anti-Su autoimmune sera were identified 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 . 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, Canada) . The hAgo1 (clone 30344513; GenBank BC063275) and hAgo4 (clone 4373725; GenBank BF979523) cDNAs in pBluescript were purchased from Open Biosystems (Huntsville, AL, USA). For both hAgo1 and hAgo4, GC-rich regions in the 5'-untranslated region were deleted to enhance the in vitro transcription and translation (TnT) reaction. Briefly, the hAgo1 plasmid was digested with EcoRI and SmaI, purified, filled in with Klenow polymerase, and religated. Similarly, the hAgo4 plasmid was digested with NotI and NcoI, purified, and ligated with a linker containing an EcoRV cut site: 5'-GGCCGATATCGTGC-3' and 5'-CATGGCACGATATC-3'. The hAgo3 cDNA (clone CS0DB008YP10; 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'-CGATACAGTCGACGCCACCATGGAAAGCCCTGCTTTGCAACC-3' and 5'-CCAATACGGCACGACAGTC-3'. All constructs were confirmed by DNA sequencing performed by the University of Florida Interdisciplinary Center for Biotechnology Research core laboratory.
Indirect immunofluorescence (IIF) analysis was previously described . Briefly, the primary antibodies to the following proteins were used: GW182 (human serum, 1:200–1:6,000; 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 analyzed 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 vitroTnT and IP
The protocol for in vitro TnT and IP was previously described . TnT reactions were performed in the presence of [35S]-methionine. The IPs of hAgo1-4 and Dicer TnT products were performed using 0.5 M NaCl NET/NP40 buffer (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 0.3% Nonidet P-40) and NET2+F buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet 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 previously [5, 23].
Results and Discussion
The Su antigen localizes to cytoplasmic structures identified as GWBs
Autoantibodies to the Su antigen were initially identified and characterized in 1984 and reported to be specifically associated 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 .
The Su antigen is biochemically similar to hAgo2
Human anti-Su autoimmune sera recognize Dicer
Serological characteristics of anti-Su autoimmune sera
Serological features of human and mouse anti-Su sera
K562 cell extract IP
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)
In summary, these data identify the Su autoantigen as a macromolecular complex closely associated with the GWB structure and the RNAi pathway. Furthermore, these data demonstrate that anti-Su autoantibodies target key components 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 members 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 significant 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 clinical significance of anti-Su autoantibodies is not apparent, it is intriguing to speculate that viruses may promote the development of autoimmunity via their association with components of the RNAi pathway. Given that RNAi is linked to the interferon system , viral suppressors inhibit RNAi [29, 30], viruses encode miRNAs , and virus-like particles associate with GWB/P-body components , it is not surprising that GWBs and thus RNAi protein and/or nucleic acid components develop into targets of autoimmunity.
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 . 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.
= double-stranded RNA
= ethylenediaminetetraacetic acid
= GW body
= indirect immunofluorescence
= polymerase chain reaction
= RNA-induced silencing complex
= RNA interference
= short interfering RNA
= systemic lupus deerythematosus
= transcription and translation
This work was supported in part with resources and the use of facilities at the Malcom Randall VA Medical Center, Gainesville, FL, National Institutes of Health grants AI39645, AI44074, AI47859, AR07603, AR40391, AR42455, AR44731, AR50661, AR51766,, and M01R00082, State of Florida funds to the Center for Autoimmune Diseases, 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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