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Fig. 6 | BMC Biology

Fig. 6

From: RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

Fig. 6

In vitro ubiquitination shows that RNA is important for TRIM25 E3 ubiquitin ligase activity. a Domain architecture of wild-type TRIM25, TRIM25 deletions mutants, and TRIM25 K117R mutant. b T7-TRIM25, but not T7-TRIM25ΔRDB, is efficiently ubiquitinated in vitro. Ubiquitination assays were performed on immunoprecipitated T7-TRIM25 or T7-TRIM25ΔRDB from HeLa cell extracts in the presence of recombinant purified 6xHis-Ube2D3, UBE1, and ubiquitin. Lanes 1 and 2 show immunoprecipitated T7-tagged proteins. Lanes 3–5 show incubation of T7-TRIM25 with 6xHis-Ube2D3, UBE1, and ubiquitin for 5, 30, and 60 minutes, respectively. Lane 6 represents 60 minutes’ reaction with T7-TRIM25, UBE1, and ubiquitin. Lanes 7–9 show incubation of T7-TRIM25ΔRDB with 6xHis-Ube2D3, UBE1, and ubiquitin for 5, 30, and 60 minutes, respectively. Lane 10 represents 60 minutes’ reaction with T7-TRIM25ΔRDB, UBE1, and ubiquitin. The proteins were analyzed by western blotting with anti-TRIM25 antibody. c Neither T7-TRIM25ΔRING nor T7-TRIM25K117R are efficiently ubiquitinated in vitro. Ubiquitination assays preformed on immunoprecipitated 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-TRIM25 antibody. d RNase A/T1 treatment significantly reduces efficiency of T7-TRIM25 ubiquitination. Lane 1 shows immunoprecipitated T7-TRIM25. Lanes 2 and 3 represent the 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-TRIM25, 6xHis-Ube2D3, UBE1, and ubiquitin in the absence and presence of RNases, respectively. Lanes 5 and 8 show reactions with immunoprecipitated T7-TRIM25, UBE1, and ubiquitin in the absence and presence of RNases, respectively. Lanes 6 and 9 show reactions with immunoprecipitated T7-TRIM25 in the absence and presence of RNases, respectively. The proteins were analyzed by western blotting with anti-TRIM25 antibody

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