PRMT6 inhibits K63-linked ubiquitination and promotes the degradation of IRF3 in the antiviral innate immunity of black carp Mylopharyngodon piceus
Introduction
In vertebrates, the immune system is the most effective weapon to defense against pathogenic microbes, which contains innate immunity and adaptive immunity (Akira et al., 2006). For innate immunity, after the detecting the pathogen-associated molecular patterns (PAMPs) of the invading virus or bacteria by the pathogen recognition receptors (PRRs) of host cells, a series of adaptors are activated, and subsequentially recruit and trigger downstream kinases, such as IκB kinases (IKKs), to initiate nuclear factor kappa B (NF-κB) and interferon regulatory factors (IRFs), leading to the production of interferons (IFNs) and proinflammatory cytokines finally (Heim and Thimme, 2014; Kumari et al., 2016; Fang et al., 2017; Tomac et al., 2021).
IRF3, as a master transcription factor for type I IFN production, is indispensable for the innate immune activation. IRF3 possesses an extremely conserved DNA binding domain (DBD) characterized by five tryptophan repeats in the N-terminal (Au and Pitha, 2001). The classical IRF association domain (IAD) and the serine-rich domain (SRD) are positioned at C-terminal, where the former is used to interact with other regulators (Tamura et al., 2008). In human and mammals, IRF3 is strictly regulated in multiple signaling pathways, including TLR or RLR signaling pathway. Post translational modification (PTM) of IRF3 is crucial for the regulation of this molecule, such as phosphorylation and ubiquitination. The activation of IRF3 depends heavily on its phosphorylation in different site, especially the C-terminal serine residues, which leads to its dimerization and nuclear migration (Lin et al., 1998; Heylbroeck et al., 2000). And the various kinds of ubiquitination of IRF3 have been widely shown to be involved in the regulation of IRF3-mediated IFN signaling. For instance, RAUL directly catalyzes K48-linked ubiquitination of IRF3, leading to proteasome-dependent degradation (Liu et al., 2016). The K63-linked ubiquitination at K98 enhances the activity of IRF3 in an autoimmune disease model (Lu et al., 2018).
In teleost fish, IRF3 is not as well studied as in human and mammals. The ability of IRF3 to induce IFN and NF-κB expression was demonstrated in multiple teleosts, such as dark sleeper (Odontobutis obscura), zebrafish (Danio rerio) (Sun et al., 2010; Yan et al., 2020; Wang et al., 2021). Dark sleeper (Odontobutis obscura) IRF3 was proved to regulate IFN in the antiviral immune response (Li et al., 2019). In addition to the function in IFN and ISG regulation, crucian carp (Carassius auratus L.) IRF3 was also identified as a canonical ISG (Xu et al., 2015). Consistent with its mammalian homologue, the nuclear migration of black carp IRF3 (bcIRF3) and bcIRF3-mediated interferon production were triggered by the inhibitor of NF-κB kinase ε (IKKε) (Wang et al., 2021). Zebrafish OTUD6b suppressed K63-linked ubiquitination of IRF3, leading to the negative regulation of IRF3 (Zhou et al., 2021).
Protein arginine methyltransferases (PRMTs) are named for their ability to methylate arginine residues of substrates protein (Gupta et al., 2021). As a member of PRMT family, PRMT6 has been widely studied, which is involved in diversified biological process, including epigenetic regulation of gene expression, alternative splicing, development and differentiation, DNA repair and immunity (Guccione et al., 2007; Lausen, 2013; Di Lorenzo et al., 2014; Gupta et al., 2021). PRMT6 regulates the activity of differentiation-related genes by reverse transcriptional effects, which leads to promoting or inhibiting cancer development, and the effect is not single and fixed (Chen et al., 2022). PRMT6 is also involved in immunity, it has been shown to inhibit the TBK1/IFN signaling pathway, however, it also acts as a coactivator of NF-κB (Di Lorenzo et al., 2014; Zhang et al., 2019).
In our previous data, bcPRMT6 were found to inhibit black carp TBK1 (bcTBK1)-mediated antiviral activity, however, the interaction between bcPRMT6 and bcIRF3, but not bcPRMT6 and bcTBK1, were identified, which intrigued us to explore the relationship between bcPRMT6 and bcIRF3 (Jiang et al., 2019). In this study, overexpression and knockdown of bcPRMT6 identified that bcPRMT6 functioned negatively in IFN signaling. bcPRMT6 promoted the degradation of bcIRF3 through lysosome pathway and attenuated the K63-linked ubiquitination of bcIRF3, which shed a light on the regulation of IRF3 during the innate immune activation of teleost.
Section snippets
Cells and virus
HEK293T cells, Epithelioma papulosum cyprini (EPC) cells, Ctenopharyngodon idella kidney (CIK) and M. piceus kidney (MPK) cells were maintained as the reference (Li et al., 2018). HEK293T cells were cultured at 37 °C; CIK, EPC and MPK cells were cultured at 26 °C, all with 5% CO2. HEK293T and EPC cells were transfected with PEI, while Lipomax was used to transfect MPK cells (Yang et al., 2019). CIK and EPC cells were used to produce grass carp reovirus (GCRV/strain: GCRV106) and spring viremia
Overexpression of bcPRMT6 down-regulated bcIRF3-mediated signaling
As downstream transcription activator of bcTBK1, bcIRF3 functions crucially in the interferon production of black carp during the antiviral innate immune activation. Our previous data has identified that bcPRMT6 suppressed bcTBK1-mediated antiviral activity like its mammalian counterpart (Jiang et al., 2019). To investigate the role of bcPRMT6 in bcIRF3-mediated antiviral signaling, MPK cells were transfected with bcPRMT6 and/or bcIRF3, and the mRNA variation of IFNs and cytokines related genes
Discussion
As a significant part of antiviral innate immunity, RLR signaling pathway is grimly monitored by both activators and inhibitors, which either cause IFN expression to facilitate immune response or prevent abnormal IFN production to avoid autoimmune disorders accordingly (Andrejeva et al., 2004). TBK1 and IKKε, two non-canonical members of IKKs, are involved in the activation of IRF3, which plays the indispensable role in IFN production (Tanaka and Chen, 2012; Liu et al., 2015). The
Credit author statement
I have made substantial contributions to the enclosed manuscript entitled “PRMT6 inhibits K63-linked ubiquitination and promotes the degradation of IRF3 in the antiviral innate immunity of black carp Mylopharyngodon piceus”, including the conception and design of the work, analysis of data for the work; and I have drafted the work and revised it critically for important intellectual content. I have approved the final version to be published; and I agree to be accountable for all aspects of the
Declaration of Competing Interest
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of the manuscript.
I am one author signing on behalf of all co-authors of this manuscript, and attesting to the above.
Acknowledgements
This work was supported the National Natural Science Foundation of China (U21A20268, 31920103016, 32002415, 32002430), Hunan Provincial Science and Technology Department (2021NK2025), the Modern Agricultural Industry Program of Hunan Province, Hunan Provincial education Department (20A317, 2022XKQ0201), the Excellent Education Team for Postgraduate in Hunan Province, and the Fish Disease, Vaccine Research and Development Platform for Postgraduates in Hunan Province and Projects of Hunan Normal
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