Figure 3. The requirement for ATP in IFN-β promoter induction is ligand dependent.
(A–C) IFN-β induction in HEK 293T cells transfected with the indicated amount of the constitutive expression plasmid pUNO-hRIG-I containing either WT or mutant RIG-I constructs. Cells expressing the indicated construct were challenged by transfection of (A) 5′ppp14L, (B) 5′ppp30L, or (C) 5′ppp50L. (D–F) Steady state ATP hydrolysis by wild type and mutant RIG-I proteins stimulated with varying concentrations of the RNA hairpin (D) 5′ppp14L, (E) 5′ppp30L, or (F) 5′ppp50L. (G) Relative affinities for MANT-ATP binding by wild type and mutant RIG-I proteins bound in the context of 5′ppp10L, 5′ppp30L normalized to WT RIG-I bound to 5′ppp10L (Figure 1B). Plotted values are mean ± SD (n = 3).
Figure 3—figure supplement 1. RIG-I forms multimers on 5′ppp50L at high protein concentrations.
An EMSA showing RIG-I binding to 5′ppp50L. Lane 1 contains 100 nM RNA, and lanes 2–5 contain wild type RIG-I incubated with 100 nM RNA at concentrations ranging from 100 nM to 1 μM. Multimer formation is demonstrated by the slower mobility bands (denoted 2:1 and 3:1) that evolve at higher protein concentrations.
Figure 3—figure supplement 2. Assessment of RNA quality.
(A) RNA hairpins and the RNA dumbbell were run on a native polyacrylamide gel to visualize sample purity. (B) A 5′ end labeling reaction was performed on the RNA dumbbell in order to demonstrate complete ligation (see ‘Materials and methods’). A total of 2–5% incorporation of [32P-γ] ATP was observed (lower spot) based on radioactive signal retention normalized to an unwashed control (upper spot). This indicates that the ligation reaction and subsequent purification yielded 95–98% pure RNA dumbbell.