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. Author manuscript; available in PMC: 2010 Oct 1.
Published in final edited form as: Cell. 2010 Apr 16;141(2):315–330. doi: 10.1016/j.cell.2010.03.029

Figure 7. Endogenous Unanchored K63 Polyubiquitin Chains Activate the RIG-I Pathway.

Figure 7

(A) A protocol for isolating functional endogenous polyUb chains in human cells. (B) Endogenous polyUb chains bound to RIG-I can be released by heat treatment and remained functional. GST-RIG-I(N)-K172-only was expressed in HEK293T cells and isolated as described in (A), then heated for 5 minutes at the indicated temperatures. After centrifugation, the supernatant containing heat-resistant ubiquitin chains was incubated with GST-RIG-I(N) expressed and purified from E. coli (lanes 7–12). The activity of GST-RIG-I(N) was then measured by IRF3 dimerization assay. As positive controls, K63 polyUb chains were incubated with GST-RIG-I(N)-K172-only, which was then pulled down and heated in parallel experiments (lanes 1–6). endo. polyUb: endogenous polyUb. (C) Endogenous unanchored polyUb chains activate RIG-I(N). PolyUb chains associated with GST-RIG-I(N)-K172-only were captured and released at 75°C as in (B). The heat-resistant supernatant was incubated with GST-RIG-I(N) followed by IsoT treatment (lane 9), or in reverse order (lane 8). As positive controls, unanchored K63 polyUb chains were incubated with GST-RIG-I(N) and IsoT in sequential orders as indicated. In the right panel, the heat supernatant containing endogenous polyUb from HEK293T cells was incubated with or without IsoT, then analyzed by immunoblotting with a ubiquitin antibody. The arrow denotes a ~40 kDa band that is likely K63-Ub6 (see Figure S7B). (D) Similar to (C), except that the supernatant containing endogenous polyUb chains were treated with CYLD. The ubiquitin chains were detected with a ubiquitin antibody or another antibody specific for the K63 linkage of ubiquitin chains. (E) siRNA oligos targeting GFP (control), TRIM25 or CYLD were transfected into HEK293T cells, which were subsequently transfected with an expression vector encoding GST-RIG-I(N)-K172-only. Endogenous polyUb chains associated with the GST-RIG-I(N) protein were isolated as described in (A), then tested in IRF3 dimerization assay and visualized by immunoblotting with a ubiquitin antibody. The efficiency of RNAi was also confirmed by immunoblotting. (F) Potent activation of RIG-I by endogenous polyUb chains. Different amounts of heat-resistant supernatant containing endogenous polyUb were incubated with GST-RIG-I(N) to measure IRF3 dimerization. The concentration of the ubiquitin chains was estimated by semi-quantitative immunoblotting (Figure S7B). Error bars represent the variation range of duplicate experiments. (G) A proposed mechanism of RIG-I activation by RNA and polyUb (see Results and Discussion).