RAD23A negatively regulates RIG-I/MDA5 signaling through promoting TRAF2 polyubiquitination and degradation

https://doi.org/10.1016/j.bbrc.2013.01.059Get rights and content

Abstract

RIG-I/MDA5 plays a pivotal role in innate immunity by detecting intracellular double-stranded RNA (dsRNA) and activating the transcription of type I interferons and proinflammatory factors, but the exactly regulating mechanism of RIG-I/MDA5 signaling remains elusive. In this study, UbL-UBA domain containing protein RAD23A was identified as a negative regulator of RIG-I/MDA5-mediated signaling activation through a small interfering RNA (siRNA)-based screening. Knockdown of RAD23A augmented the expression of RIG-I/MDA5-mediated expression of proinflammatory cytokines and IFN-β whereas ectopic expression of RAD23A showed the converse effect. Moreover, we confirmed the interaction between RAD23A and tumor necrosis factor receptor-associated factor 2 (TRAF2), an essential mediator of RIG-I/MDA5 signaling, and found that RAD23A down-regulated TRAF2 protein level through ubiquitin–proteasome system. Therefore, this study identified RAD23A as a novel negative regulator of RIG-I/MDA5 mediated anti-virus response.

Highlights

► RAD23A negatively regulates RIG-I/MDA5 mediated signaling. ► RAD23A interacts with TRAF2 through UBA1 domain. ► RAD23A promotes TRAF2 ubiquitin-proteasome dependent degradation.

Introduction

Innate immunity detects microbial pathogen invasion through germline-encoded pattern-recognition receptors (PRRs) which recognize pathogen associated molecular patterns (PAMPs), including exogenous DNA, single-stranded (ss) RNA, double-stranded (ds) RNA and glycoproteins, etc. Viral dsRNA is recognized through two distinct pathways. Toll-like receptor 3 (TLR3) detects dsRNA phagocytosed in endosomes; helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene-5 (MDA-5) detect cytoplasmic dsRNA generated during viral replication [1], [2]. Activation of RIG-I/MDA5 leads to the assembly of a signaling complex composed of MAVS, tumor necrosis factor (TNF) receptor-associated factors (TRAFs), TANK-binding kinase 1 (TBK1), inhibitor of nuclear factor κB kinase ε (IKKε), etc. [3], [4], [5], [6]. Then, transcription factors NF-κB, IRF3/IRF7, and ATF/c-Jun will be activated and promote the expression of proinflammatory cytokines such as TNF-α, interleukin-6 (IL-6) and type I interferons (IFN-I) [7].

Immune signaling pathways have to be tightly regulated to insure a successful immune response against viral infections; otherwise, aberrant IFNs or proinflammatory cytokines expression would be destructive rather than protective. Recent works have shown that RIG-I/MDA5 mediated NF-κB activation is transducted by TRADDs, TRAFs, RIP1, and FADD, which are also essential mediators of other signaling pathways such as TNF receptor signaling and TLRs signaling. The RIG-I/MDA5 signaling to IRFs has been reported to proceed through TRADD, TRAFs, TANK, NAP1, NEMO, TBK1, and IKKε [6]. However, many of the details in the regulation of RIG-I/MDA5 signaling need to be clarified. The mitochondrial protein MAVS [3], [8], which directly associates with RIG-I/MDA5, is believed to be the central component of RIG-I/MDA5 signaling complex. MAVS contains TRAF2 and TRAF6 binding motifs and is reported to interact with both proteins [3], [5]. Mikkelsen et al., reported that TRAF2 is essential for phosphorylation of the IκB and p38 and IFN-stimulated genes expression [9]. Yoshida et al., found that TRAF6 and MEKK1 play important roles in RLR signaling to NF-κB and MAPKs. TRAF3 is indispensable for RIG-I/MDA5-mediated activation of IFN regulatory factors (IRFs) [10], [11]. Therefore, the fine-tuning of TRAFs is an important step for RIG-I/MDA5 signaling regulation.

Ubiquitination plays a key regulatory role in diverse cellular events, including innate immunity signaling [12], [13]. The best known function of ubiquitination is to target protein degradation by the proteasome through covalent attachment of polyubiquitin chains (Lys-48 linked), which is a major mechanism by which cells regulate the abundance of particular proteins [14]. Whereas K63-linked polyubiquitination can direct other outcomes, such as DNA repair, protein recruitment, protein kinase activation and chromatin dynamics [15]. UBDs (ubiquitin binding domains) are a collection of modular protein domains that non-covalently bind to ubiquitin [16], [17]. UBDs, such as the ubiquitin-associated domain (UBA), ubiquitin-interacting motif (UIM), A20 zinc finger (ZnF) domain influence various cellular events through binding to and regulating ubiquitinated proteins. Increasing evidences showed that UBD containing proteins play essential roles in regulating signaling in immune system [18], [19].

To identify potential roles of UBD containing proteins in RIG-I/MDA5-mediated NF-κB and IRFs signaling, we used siRNAs to knockdown various UBD containing proteins in human embryonic kidney (HEK) 293 cells. Further, to mimic the viral RNA infection, polyinosine:polycytidine acid [poly(I:C)], the RIG-I/MDA5 ligand, was transfected into cells. The NF-κB activation and IFN-β production were determined by luciferase reporter assay. We identified RAD23A but not its homolog RAD23B, as a negative regulator of RIG-I/MDA5 signaling, and found that RAD23A was involved in RIG-I/MDA5 signaling through binding to TRAF2. Furthermore, we found that RAD23A down-regulate TRAF2 by promoting its degradation through ubiquitin–proteasome system. Therefore, our finding indicated that RAD23A could be a novel regulator of antivirus immunity.

Section snippets

siRNA based screening

For screening assay, HEK293 cells were transfected with NF-κB and IFN-β luciferase reporter for 6 h and re-plated in 96-well format. Cell density should be 50–60% confluent after 12 h and the indicated siRNA (20 nM) smart pool (Thermo Scientific DharmaFECT) was transfected. 48 h later, cells were treated with Lipofectamine 2000 (Invitrogen) packed poly(I:C) (5 mg/ml, Invivogen) for 10 h, then NF-κB and IFN-β reporter activity were measured with Dual-Luciferase Reporter Assay System (Promega).

Transfection

HEK293

Knockdown of RAD23A augments RIG-I/MDA5 mediated signaling activation

RIG-I/MDA5 recognizes viral RNAs in cytoplasm and triggers signal transduction cascades that finally lead to the production of type I IFNs and proinflammatory cytokines to suppress viral replication and assembly. This signaling is closely regulated, but the exact mechanism of this regulation, however, remains elusive. To explore potential roles of UBD containing proteins in dsRNA sensor RIG-I/MDA5 mediated signaling, we used a siRNA library to knockdown human genes encoding proteins containing

Conclusions

RIG-I/MDA5 recognizes viral RNAs in cytoplasm and triggers signal transduction cascades that finally lead to the production of type I IFNs and proinflammatory cytokines to suppress viral replication and assembly. This signaling is closely regulated, but the exact mechanism of this regulation, however, remains elusive. In our study, RAD23A, an UBD domain containing protein, was identified as a novel regulator of RIG-I/MDA5 signaling in a siRNA-based screening. RAD23A negatively regulates

Acknowledgments

This work is supported by The Natural Science Foundation of China (30970593).

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