Fig. 8
From: RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination
RNA is a scaffold for TRIM25-mediated ubiquitination of ZAP. a T7-TRIM25 but not T7-TRIM25ΔRDB efficiently ubiquitinated T7-ZAP protein in vitro. Ubiquitination assays were performed on immunoprecipitated T7-ZAP with T7-TRIM25 or T7-TRIM25ΔRDB from HeLa cell extracts in the presence of recombinant purified 6xHis-Ube2D3, UBE1, and ubiquitin (the concentrations of proteins from the ubiquitin pathway was higher (hc) than in the experiments presented in Fig. 6). Lanes 1 and 2 show immunoprecipitated T7-tagged proteins. Lanes 3–5 show incubation of T7-TRIM25 and T7-ZAP with 6xHis-Ube2D3, UBE1, and ubiquitin for 5, 30, and 60 minutes, respectively. Lane 6 represents 60 minutes’ reaction with T7-TRIM25, T7-ZAP, UBE1, and ubiquitin. Lanes 7–9 show incubation of T7-TRIM25ΔRDB and T7-ZAP with 6xHis-Ube2D3, UBE1, and ubiquitin for 5, 30, and 60 minutes, respectively. Lane 10 represents 60 minutes’ reaction with T7-TRIM25ΔRDB, T7-ZAP, UBE1, and ubiquitin. The proteins were analyzed by western blotting with anti-ZAP and anti-TRIM25 antibodies. b T7-TRIM25ΔRING but not T7-TRIM25K117R are defective in ZAP ubiquitination. Ubiquitination assays preformed on immunoprecipitated T7-ZAP with T7-TRIM25, T7-TRIM25ΔRING, or T7-TRIM25K117R from HeLa cell extracts in the presence of recombinant purified 6xHis-Ube2D3, UBE1, and ubiquitin. Lanes 1–3 show immunoprecipitated T7-tagged proteins. Lanes 4, 6, and 8 represent 60 minutes’ reaction with T7-tagged immunoprecipitated proteins, 6xHis-Ube2D3, UBE1, and ubiquitin. Lanes 5, 7, and 9 represent 60 minutes’ reaction with T7-tagged immunoprecipitated proteins, UBE1, and ubiquitin. The proteins were analyzed by western blotting with anti-ZAP and anti-TRIM25 antibodies. c RNase A/T1 treatment substantially reduces efficiency of TRIM25-mediated ubiquitination of ZAP. Lane 1 shows immunoprecipitated T7-TRIM25 and T7-ZAP. Lanes 2 and 3 represent control 60 minutes’ incubation of HeLa cell extracts in the absence and presence of RNases, respectively. Lanes 4 and 7 show reactions with immunoprecipitated T7-ZAP, T7-TRIM25, 6xHis-Ube2D3, UBE1, and ubiquitin in the absence and presence of RNases, respectively. Lanes 5 and 8 show reactions with immunoprecipitated T7-ZAP, T7-TRIM25, UBE1, and ubiquitin in the absence and presence of RNases, respectively. Lanes 6 and 9 show reactions with immunoprecipitated T7-ZAP and T7-TRIM25 in the absence and presence of RNases, respectively. The proteins were analyzed by western blotting with anti-ZAP and anti-TRIM25 antibodies. d Ubiquitination of T7-ZAP is mediated by T7-TRIM25. Control incubation of immunoprecipitated T7-ZAP with 6xHis-Ube2D3, UBE1, and ubiquitin shows lack of ubiquitination. Lane 1 shows immunoprecipitated T7-ZAP. Lanes 3–5 show incubation of T7-ZAP with 6xHis-Ube2D3, UBE1, and ubiquitin for 5, 30, and 60 minutes, respectively. The proteins were analyzed by western blotting with anti-ZAP antibody