RNF122 targets STING for ubiquitination at residues K95, K117, and K155 to regulate antiviral responses in a teleost fish

Abstract

Ring finger protein 122 (RNF122), an E3 ubiquitin ligase, orchestrates antiviral immune responses in mammals by targeting retinoic acid-inducible gene 1 and melanoma differentiation-associated gene 5 for ubiquitination. However, its functional relevance in teleosts has yet to be clearly defined, particularly regarding the identification of substrate-specific regulatory sites. This study characterized RNF122 from mandarin fish ( Siniperca chuatsi), termed scRNF122, and investigated its regulatory impact on stimulator of interferon genes (STING)-mediated antiviral signaling. Results showed that scRNF122 expression was up-regulated in response to mandarin fish ranavirus (MRV) infection, and its overexpression suppressed scSTING-mediated interferon (IFN) production and enhanced MRV replication. Co-immunoprecipitation confirmed a direct interaction between scRNF122 and scSTING. Functional assays demonstrated that scRNF122 facilitated scSTING degradation through the ubiquitin-proteasome pathway, a process impeded by MG132 treatment. Ubiquitination analyses of various scSTING mutants revealed that scRNF122 catalyzed scSTING ubiquitination at K95, K117, and K155 residues. Moreover, scRNF122 significantly impaired scSTING-dependent antiviral responses by engaging negative regulatory elements within the signaling cascade. Overall, scRNF122 was identified as a negative modulator of STING-mediated IFN signaling in mandarin fish, diminishing STING-dependent antiviral activity and promoting its degradation via the ubiquitin-proteasome pathway at lysine residues K95, K117, and K155. These findings provide mechanistic insight into the post-translational control of STING in teleosts and establish a foundation for future investigations into antiviral immune regulation.

Keywords: Innate immunity; Interferon; RNF122; STING; Ubiquitination.

Figure 1

Characterization of scRNF122 and its role in promoting viral replication A: Predicted domain structure of scRNF122. B: Phylogenetic analysis of RNF122 sequences from representative vertebrate species (GenBank accession numbers are provided in Supplementary Table S3). C: Tissue distribution of scRNF122 protein expression in healthy mandarin fish. Total RNA was extracted from blood, brain, liver, spleen, fin, gill, intestine, heart, skin, head kidney, and muscle samples obtained from three healthy mandarin fish, with scRNF122 expression quantified by RT-qPCR. D: Induction of scRNF122 in MFF-1 cells following MRV infection (multiplicity of infection (MOI) of 0.1), with scRNF122 mRNA expression determined by RT-qPCR and normalized to β-actin. Cells were transfected with Myc-scRNF122 or pCMV-Myc for 24 h, then infected with MRV and harvested at indicated time points. E–I: RT-qPCR quantification of scRNF122, scIFN-h, mrvMCP, mrvICP18, and mrvDNA-Poly expression in MRV-infected cells at indicated times. Expression values were normalized to the lowest-expressing group, set to 1. J: Viral titers determined by endpoint dilution assay in 96-well culture plates. K: Detection of mrvORF097L protein levels by WB analysis. L: Densitometric quantification of mrvORF097L normalized to GAPDH. Vertical bars represent ±SD (n=3). ^**: P<0.01.

Figure 2

scRNF122 inhibits scSTING-induced IFN signaling and physically interacts with scSTING A: Relative IFN-β-luc activity was measured in cells co-transfected with Flag-scSTING and Myc-scRNF122. Cells transfected with pCMV-Myc alone served as the negative control. B: MFF-1 cells were co-transfected with pRL-TK and IFN-β-luc reporter plasmids, along with scRNF122 and scSTING expression plasmids in 24-well plates. Luciferase activity was assessed 36 h post-transfection. Vertical bars represent ±SD (n=3). ^**: P<0.01. C–F: mRNA expression levels of scIFN-h (C), scMx (D), scISG15 (E), and scTNF-α (F) were quantified by RT-qPCR 24 h after co-transfection with scSTING and scRNF122 or vector. Data represent three independent experiments (n=3). ^**: P<0.01. G, H: FHM cells were transfected with corresponding plasmids or empty vectors, followed by Co-IP and WB analyses to examine the interaction between scRNF122 and scSTING. IB: Immunoblotting; IP: Immunoprecipitation. I: FHM cells were co-transfected with pCMV-Myc-scRNF122 or empty vector, pCMV-Flag-scSTING(1–140), and pCMV-Flag-scSTING(141–417) plasmids. Co-IP and WB analyses were performed to map interaction regions.

Figure 3

scRNF122 mediates scSTING degradation via ubiquitination targeting the 94–180 amino acid region A: WB analyses were used for detection. FHM cells were transfected with pCMV-Myc-scRNF122 or an empty vector alongside pCMV-Flag-scSTING plasmids. B: FHM cells were seeded in 6-well plates, incubated overnight, and co-transfected with 2 μg Flag-scSTING and Myc-scRNF122 (0.5, 1, and 2 μg), After 24 h, lysates were immunoblotted with anti-Flag, anti-Myc, and anti-GAPDH antibodies. C: Effects of MG132, 3-MA, CQ, and NH₄Cl on scRNF122-induced scSTING degradation were assessed by treating FHM cells in 6-well plates with 2 μg of Flag-scSTING and 2 μg of Myc-scRNF122. At 24 h post-transfection (hpt), cells were treated with DMSO or respective inhibitors (MG132: 20 μmol/L, 3-MA: 5 μmol/L, CQ: 10 μmol/L, NH₄Cl: 20 μmol/L). After 12 h, lysates were immunoblotted using anti-Flag, anti-Myc, and anti-GAPDH antibodies. D: FHM cells were transfected and treated with increasing MG132 concentrations (1, 10, and 20 μmol/L), and a negative control (MG132). Lysates were immunoblotted as before. E: Ubiquitination of scSTING was assessed in FHM cells co-expressing Flag-scSTING, Ha-ubiquitin, and either Myc-scRNF122 (lane 2) or empty vector (lane 1). Flag-scSTING was immunoprecipitated with anti-Flag, and poly-ubiquitin chains were detected with anti-Ha. F: Positions of lysine (K) residues in truncated STING variants. G, H: FHM cells were seeded overnight in 6-well plates and co-transfected with 2 μg of either Flag-scSTING(1–180) or Flag-scSTING(181–417), along with Myc-scRNF122 at concentrations of 0.5, 1, or 2 μg. After 24 h, lysates were immunoblotted using anti-Flag, anti-Myc, and anti-GAPDH antibodies. I: Ubiquitination of scSTING fragments was assessed by co-transfecting FHM cells with Myc-scRNF122, Ha-ubiquitin, and either Flag-scSTING(1–180) or Flag-scSTING(181–417). Flag-scSTING was immunoprecipitated using anti-Flag, and poly-ubiquitin chains were detected with anti-Ha. J–N: Similarly, FHM cells were seeded and co-transfected as in C, but with Flag-scSTING(1–140), Flag-scSTING(1–134), Flag-scSTING(1–121), Flag-scSTING(1–116), or Flag-scSTING(1–94). Lysates were analyzed by immunoblotting as described above.

Figure 4

scRNF122 targets scSTING for ubiquitination at K95, K117, and K155 A: Schematic of scSTING mutants used in this study. B, C: FHM cells were co-transfected with 2 μg of Flag-scSTINGCKR/Flag-scSTING(1–180)KR and Myc-scRNF122 (0.5, 1 and 2 μg) for 24 h. Lysates were then subjected to immunoblotting with anti-Flag, anti-Myc, and anti-GAPDH Abs. D–H: FHM cells were co-transfected with 2 μg of various Flag-tagged scSTING mutants, including scSTINGCKR(1–161)155K, scSTINGCKR(1–141)135K, scSTINGCKR(1–128)122K, scSTINGCKR(1–123)117K, and scSTINGCKR(1–101)95K, along with Myc-scRNF122 (0.5, 1, and 2 μg) for 24 h. Lysates were then immunoblotted with anti-Flag, anti-Myc, and anti-GAPDH Abs. I: Additional schematic of scSTING mutants employed in this study. J–O: FHM cells were seeded and co-transfected as in B, but with specific scSTINGCKR mutants: scSTINGCKR20K, scSTINGCKR95K, scSTINGCKR117K, scSTINGCKR122K, scSTINGCKR135K and scSTINGCKR155K. Lysates were processed for immunoblotting. P: L02 cells were seeded and co-transfected as in B, but with hsSTING. Lysates were analyzed as described above.

Figure 5

scRNF122 ubiquitinates specific lysine residues (K95, K117, and K155) of scSTING A: Schematic representation of scSTING mutants used in this study. B: FHM cells were co-transfected with 2 μg of Flag-scSTING mutants (K95R, K117R, K155R, and K95/117/155R) and increasing amounts of Myc-tagged scRNF122 (0.5, 1, and 2 μg) for 24 h. Cell lysates were then subjected to immunoblotting with anti-Flag, anti-Myc, and anti-GAPDH antibodies. C: Ubiquitination of scSTING mutants (K95R, K117R, K155R, and K95/117/155R) was assessed. FHM cells were co-expressed with Myc-scRNF122, Ha-ubiquitin, and either Flag-scSTING, Flag-scSTINGK95R, Flag-scSTINGK117R, Flag-scSTINGK155R, or Flag-scSTINGK95/117/155R. Flag-scSTING was immunoprecipitated using anti-Flag, and poly-ubiquitin chains were detected with anti-Ha. D: MFF-1 cells were co-transfected in 24-well plates with reporter plasmids pRL-TK and IFN-β-luc, along with plasmids encoding scRNF122 or pCMV-Myc and scSTING mutants. Luciferase activity was measured 36 h post-transfection. E: Protein amino acid alignment revealed conserved amino acid sequences and distribution of lysine sites in STING across different species. Accession numbers for these sequences are provided in Supplementary Table S3. Arrows indicate lysine residues at positions 95, 117, and 155 in scSTING. Asterisk (^*) represents fish belonging to the order Perciformes.

Figure 6

scRNF122 inhibits scSTING-mediated antiviral activity A–F: MFF-1 cells were infected with MRV and harvested at the indicated time points for RT-qPCR, WB, and TCID₅₀ analyses. Expression levels of scRNF122 (A), scSTING (B), scIFN-h (C), mrvMCP (D), mrvICP18 (E), and mrvDNA-Poly (F) in MRV-infected cells at indicated times. For RT-qPCR, the baseline was set as the group with the lowest relative expression among all groups, assigned a value of 1. Each group represented different time points after viral infection. G: Virus titers were determined using the endpoint dilution method. H: WB analysis was performed to assess levels of the viral protein mrvORF097L, with densitometric quantification normalized to GAPDH. Left two lanes represent samples collected at 24 hpi, while right two lanes indicate samples collected 48 hpi. Vertical bars represent ±SD (n=3). ^**: P<0.01.

Figure 7

Schematic representation of RNF122-mediated negative regulation of STING signaling in mandarin fish scRNF122 interacted with scSTING and catalyzed ubiquitination at lysine residues K95, K117, and K155. This post-translational modification targeted scSTING for proteasomal degradation, thereby preventing IFN signaling. Through targeted degradation of scSTING, scRNF122 impaired the host antiviral response and promoted viral replication.