RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response

Abstract

Protein modifications by ubiquitin and small ubiquitin-like modifier (SUMO) play key roles in cellular signaling pathways. SUMO-targeted ubiquitin ligases (STUbLs) directly couple these modifications by selectively recognizing SUMOylated target proteins through SUMO-interacting motifs (SIMs), promoting their K48-linked ubiquitylation and degradation. Only a single mammalian STUbL, RNF4, has been identified. We show that human RNF111/Arkadia is a new STUbL, which used three adjacent SIMs for specific recognition of poly-SUMO2/3 chains, and used Ubc13-Mms2 as a cognate E2 enzyme to promote nonproteolytic, K63-linked ubiquitylation of SUMOylated target proteins. We demonstrate that RNF111 promoted ubiquitylation of SUMOylated XPC (xeroderma pigmentosum C) protein, a central DNA damage recognition factor in nucleotide excision repair (NER) extensively regulated by ultraviolet (UV)-induced SUMOylation and ubiquitylation. Moreover, we show that RNF111 facilitated NER by regulating the recruitment of XPC to UV-damaged DNA. Our findings establish RNF111 as a new STUbL that directly links nonproteolytic ubiquitylation and SUMOylation in the DNA damage response.

Figure 1.

Human RNF111 binds to poly-SUMOylated proteins via an N-terminal SIM region. (A) Schematic of human RNF111/Arkadia. The RING domain, two putative NLSs (Episkopou et al., 2001), and three SUMO-interacting motifs (SIMs; top), conserved in higher vertebrates (bottom), are shown. Core hydrophobic SIM residues are highlighted in green. (B) Amino acid substitutions (highlighted in red) in the RNF111 SIM region to disrupt its SUMO-binding ability (*SIM). (C) S-FLAG-Strep–tagged RNF111 (SFS-RNF111) proteins expressed in U2OS cells were purified on Strep-Tactin Sepharose, incubated with purified SUMO2 or poly-SUMO2 (3–8), and washed extensively. Bound complexes were immunoblotted with the SUMO2 antibody. WCE, whole-cell extract. (D) HeLa cells stably expressing FLAG-SUMO isoforms were transfected with Strep-HA-RNF111 plasmids as indicated. Whole-cell extracts were subjected to Strep-Tactin pull-down and immunoblotting with the FLAG antibody. (E) Plasmon surface resonance analysis of poly-SUMO2 binding kinetics of RNF111 fragments spanning the SIMs. Data shown are from a single representative experiment out of three repeats. MM, molecular mass.

Figure 2.

RNF111 has STUbL activity in the presence of Ubc13–Mms2. (A) MS-based analysis of RNF111-interacting proteins. U2OS and U2OS/GFP-RNF111 cells were grown in light and heavy SILAC medium, respectively. GFP-RNF111 and associated proteins enriched on GFP-Trap resin were analyzed by MS. Plot shows z scores (from SILAC heavy/light ratios) and total intensity of identified proteins. RNF111, Ubc13 (UBE2N), and Mms2 (MMS2) are highlighted. See also Fig. S1 (A and B). (B) U2OS cells were cotransfected with indicated combinations of GFP-RNF111 and Strep-HA-Ubc13 plasmids. Whole-cell extracts (WCE) were subjected to Strep-Tactin pull-down followed by immunoblotting with GFP and HA antibodies. (C) U2OS cells transfected with nontargeting (control [CTRL]) or RNF111 siRNAs were collected 72 h later and processed for immunostaining (top) or immunoblot (bottom) with RNF111 antibody. Asterisk indicates a nonspecific band. Bar, 10 µm. (D) Extracts of U2OS cells sequentially transfected with RNF111 siRNA and S-FLAG-Strep–tagged RNF111 (SFS-RNF111) plasmids were subjected to Strep-Tactin pull-down. Bound complexes were incubated with ubiquitylation reaction mixture containing E1, Ubc13–Mms2 complex, and HA-ubiquitin as indicated and washed extensively, and RNF111 E3 ligase activity was analyzed by immunoblotting with the HA antibody. (E) As in D, except that ubiquitylation reactions were performed in the presence or absence of poly-SUMO2 (3–8) chains followed by immunoblotting with HA and SUMO2 antibodies. MM, molecular mass.

Figure 3.

RNF111 promotes UV-induced ubiquitylation of XPC. (A) U2OS or U2OS/Strep-HA-ubiquitin cells transfected with control (−) or RNF111 siRNAs were exposed or not exposed to UV and collected 1 h later, and XPC ubiquitylation was analyzed by immunoblotting Strep-Tactin pull-downs of whole-cell extracts (WCE) with the XPC antibody. (B) HeLa/FLAG-SUMO2 cells transfected with control (−) or RNF111 siRNAs and left untreated or induced to express FLAG-SUMO2 by addition of doxycycline (DOX) were exposed or not exposed to UV and collected 1 h later. Cells were lysed under denaturing conditions, and XPC SUMOylation was analyzed by immunoblotting of FLAG IPs with XPC antibody. (C) U2OS/Strep-HA-ubiquitin cells transfected with empty vector (−) or FLAG-RNF111 plasmid were exposed or not exposed to UV and collected 1 h later. XPC ubiquitylation was analyzed as in A. (D) XPC ubiquitylation in U2OS/Strep-HA-ubiquitin cells depleted of RNF111 or Ubc13 was analyzed as in A. Ubc13 knockdown efficiency is shown in Fig. S3 D. (E) Extracts of U2OS/GFP-RNF111 cells collected at the indicated times after UV radiation were subjected to GFP IP followed by immunoblotting with XPC antibody. (F) Extracts of U2OS cells incubated with or without MG132, exposed to UV 30 min later, and collected at the indicated times after UV were analyzed by immunoblotting with the RNF111 antibody. Asterisks denote a nonspecific band. MM, molecular mass.

Figure 4.

RNF111 ubiquitylates XPC in a SUMOylation-dependent manner. (A) Outline of in vitro SUMO-binding and STUbL assays. XPC-GFP expressed in U2OS cells was immunopurified on GFP-Trap resin and subjected to in vitro SUMOylation. After washing, the XPC-GFP–containing beads were incubated with extracts of cells transfected or not transfected with S-FLAG-Strep-RNF111 (SFS-RNF111) constructs, washed again, and processed for immunoblotting (IB) of bound SFS-RNF111 with FLAG antibody (i) or subjected to in vitro ubiquitylation followed by washing and immunoblotting with the HA antibody to analyze ubiquitin ligase activity (ii). (B) SUMOylation-dependent binding of RNF111 to XPC, analyzed as described in A. (C) Analysis of SUMOylation-dependent XPC ubiquitylation by RNF111 was performed as described in A. MM, molecular mass.

Figure 5.

RNF111 promotes NER by regulating XPC recruitment to UV-damaged DNA. (A) UDS of the indicated MEF cell lines, determined by EdU incorporation for 3 h after exposure to 16 J/m² UV-C. Error bars indicate SDs of three independent experiments. (B) Cells stably expressing XPC-GFP were transfected with indicated siRNAs and locally exposed to laser-induced UV-C damage. XPC-GFP fluorescence intensity at the damaged area relative to predamage intensity was recorded in time using live-cell confocal imaging (mean of three independent experiments, n = 8 cells per experiment, ±SD). (C) As in B, except that cells were transfected with control (CTRL) or DDB2 siRNA. Results of a representative experiment (n = 8 cells per sample, ±SEM) are shown.