Figure 4. RIG-I’s ATP hydrolysis enhances RNA end recognition and removes RIG-I from RNA stems.

(A) Quantification of electrophoretic mobility shift assays of RIG-I or RIG-I E373Q incubated with 24mer dsRNA in presence or absence of ATP, ADP or ADP·BeF₃ (compare with Figure 4—Figure supplement 1B). (B) Fluorescence anisotropy changes measured by titrating RIG-I or RIG-I E373Q in presence or absence of ATP into solutions containing fluorescently labeled 12mer dsRNA. (C) Fluorescence anisotropy changes measured by titrating RIG-I or RIG-I E373Q in presence or absence of ATP into solutions containing fluorescently labeled 12mer ppp-dsRNA. (D) Quantification of electrophoretic mobility shift assays of RIG-I, RIG-I E373Q or RIG-I T347A, E373Q incubated with an RNA hairpin derived from helix A of the human ribosome expansion segment 7L (ES hairpin) in presence or absence of ATP, ADP or ADP·BeF₃ (compare with Figure 4—Figure supplement 1C). All binding curves were fitted using the LL.2 function of the R drc package (Cedergreen et al., 2005). n=3-6, error bars represent mean values ± standard deviation.

DOI: http://dx.doi.org/10.7554/eLife.10859.011

Figure 4—figure supplement 1. Design of the ribosomal expansion segment derived hairpin RNA, EMSA raw figures and control experiments with RIG-I C268F SMS mutant.

(A) RIG-I E373Q binding site at ES7L-A was used to design a 60b hairpin RNA (ES hairpin). RNA secondary structure was determined with the RNAfold webserver (Gruber et al., 2008). (B) Electrophoretic mobility shift assays of RIG-I or RIG-I E373Q incubated with 24mer dsRNA. Complexes were pre-formed at 37 °C for 20 min, separated on agarose gels and stained with GelRed. Free RNA bands were quantified using ImageJ. Protein concentrations (from left to right): 0, 0.1 µM, 0.3 µM, 0.5 µM, 0.7 µM, 1 µM, 1.5 µM and 2 µM. *: unbound RNA, **: protein:RNA complexes. (C) Electrophoretic mobility shift assays of RIG-I, RIG-I E373Q or RIG-I C268F incubated with ES hairpin RNA. Complexes were pre-formed, separated and stained as in panel B. Protein concentrations (from left to right): 0, 0.5 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5 µM and 10 µM. *: unbound RNA, **: protein:RNA complexes.

DOI: http://dx.doi.org/10.7554/eLife.10859.012