The autoinhibitory CARD2-Hel2i Interface of RIG-I governs RNA selection

Abstract

RIG-I (Retinoic Acid Inducible Gene-I) is a cytosolic innate immune receptor that detects atypical features in viral RNAs as foreign to initiate a Type I interferon signaling response. RIG-I is present in an autoinhibited state in the cytoplasm and activated by blunt-ended double-stranded (ds)RNAs carrying a 5' triphosphate (ppp) moiety. These features found in many pathogenic RNAs are absent in cellular RNAs due to post-transcriptional modifications of RNA ends. Although RIG-I is structurally well characterized, the mechanistic basis for RIG-I's remarkable ability to discriminate between cellular and pathogenic RNAs is not completely understood. We show that RIG-I's selectivity for blunt-ended 5'-ppp dsRNAs is ≈3000 times higher than non-blunt ended dsRNAs commonly found in cellular RNAs. Discrimination occurs at multiple stages and signaling RNAs have high affinity and ATPase turnover rate and thus a high katpase/Kd. We show that RIG-I uses its autoinhibitory CARD2-Hel2i (second CARD-helicase insertion domain) interface as a barrier to select against non-blunt ended dsRNAs. Accordingly, deletion of CARDs or point mutations in the CARD2-Hel2i interface decreases the selectivity from ≈3000 to 150 and 750, respectively. We propose that the CARD2-Hel2i interface is a 'gate' that prevents cellular RNAs from generating productive complexes that can signal.

Figure 1.

Structural model of RIG-I activation and schematics of RIG-I constructs used in this study. (A) Autoinhibited RNA free RIG-I modeled using structures of full length duck RIG-I (PDB ID: 4a2w) and duck RIG-I RD (PDB ID: 4a2v). Active dsRNA bound RIG-I modeled using structures of helicase-RD (PDB ID: 3tmi) and duck RIG-I CARD domains (PDB ID: 4a2w). (B) Schematic of human-RIG-I full length domains and the different deletion and mutant constructs used in this study. Models were generated using Pymol (Version 1.7.4., Schrödinger, LLC).

Figure 2.

K_d values of full-length RIG-I complexes with blunt-end and non-blunt ended dsRNA in the absence and presence of ATP hydrolysis. (A–B) Fluorescence anisotropy of 5′ fluorescein labeled dsRNA with 5′ppp or 5′OH (2 nM) was measured after addition of increasing amounts of RIG-I. The dissociation constant (K_d) was determined from fitting the data to Equations (1) and (2) (Experimental methods). (C–D) The ATPase turnover rates of RIG-I (5 nM) was measured with increasing concentration of 5′ppp or 5′OH RNA. The data were fit to the quadratic equation to obtain the apparent dissociation constants (K_d,_app). (E) The K_d,_app of RIG-I complexes with the indicated hairpin RNAs were obtained from the ATPase based titrations at 37°C in Buffer A. (F) The K_d from the anisotropy assay (black bars) and K_d,_app from the ATPase assay at 15°C (red bars) for RIG-I complexes with the indicated RNAs. Errors are standard errors from the fittings.

Figure 3.

K_d values of the C-terminal RIG-I RD complexes with blunt-end and non-blunt ended dsRNAs. (A–B) Fluorescence intensity of 3′ fluorescein labeled dsRNA with 5′ppp or 5′OH (2 nM) was measured after addition of increasing concentration of the RD protein. The data were fit to Equation (3) to obtain the dissociation constant (K_d) values. (C–E) Fluorescence anisotropy of 5′ fluorescein labeled overhang dsRNAs (40 nM) was measured with increasing concentrations of RD and data were fit to Equations (1) and (2) to obtain the K_d values. (F) The bar chart compares the K_d values of RD and RIG-I complexes with the indicated RNAs. Standard errors from fitting are shown.

Figure 4.

Loss of RNA binding selectivity upon removal of CARDs or mutation in the CARD2-Hel2i interface. (A) Helicase-RD (5 nM) was titrated with increasing concentrations of 5′OH RNA (black circles) or 5′ppp RNA (red inverted triangles) and the ATPase turnover rates were measured at 15°C in Buffer A. The binding curves show stoichiometric 1:1 binding of Helicase-RD and RNA. (B) Bar Chart compares the apparent dissociation constant K_d,app of Helicase-RD (blue bars) for the various RNAs are shown in comparison to RIG-I (gray bars). The K_d of Helicase-RD for 5′ppp RNA was determined from the off and on rates. Standard errors from the fitting are shown. (C) The CARD2 (blue) and Hel2i (yellow) interface residues in duck RIG-I and the corresponding residues inhuman RIG-I (in parentheses) are shown. CARD2 residues R109 and L110 interact with Hel2i residues E531 and F539, respectively. (D) Bar Chart compares the K_d,_app values of R109A/L110A RIG-I (green bars) complexes with indicated RNAs are shown in comparison to RIG-I (gray bars). Standard errors from fitting are shown (also see Supplementary Table S3).

Figure 5.

The ATPase activity and RNA selectivity of full-length RIG-I, Helicase-RD and R109A/L110A RIG-I mutant. (A–C) The bar chart compares the indicated RNA stimulated ATPase turnover rates of RIG-I (A) and Helicase-RD (B) and R109A/L110A (C) at 1 mM ATP and 1 μM of RNAs measured at 15°C in Buffer A. Errors from two independent experiments are shown. (D) The bar chart compares the RNA selectivity of RIG-I, Helicase-RD and R109A/L110A for the indicated RNAs normalized to the selectivity for 5′ppp RNA (Also see Supplementary Table S5).

Figure 6.

Signaling Activity of dsRNA with various end-modifications. (A) The k_atpase/RNA K_d,app of each RNA ligand (blue bars) is plotted alongside their respective IFN-β promoter response elicited in signaling assays (red bars). Error bars are SEM of data collected from quadruplicate sets. (B) Correlation between signaling and logarithm of k_atpase/K_d of RNAs.

Figure 7.

Model of RNA selectivity and RIG-I activation. RIG-I exists in the autoinhibited state in the absence of RNA binding where the CARD2 (C2, blue) is interacting with the Hel2i (yellow). PAMP and non-PAMP RNAs are sampled initially by RIG-I's C-terminal RD (red). PAMP RNAs (e.g. 5′ppp blunt-ended dsRNAs) bind with a high affinity (pathway on the left) to RD and the helicase domains (Hel1, Hel2, Hel2i), and the complex undergoes an induced-fit to disrupt the CARD2-Hel2i interactions by rotation of the Hel2-Hel2i subdomains, which results in new interactions with the dsRNA. These complexes have high affinity and high ATPase activity, with the potential to and undergo downstream events like ubiquitination and/or oligomerization, which ultimately leads to signaling. The RD and helicase binds weakly to non-PAMP RNAs (e.g. dsRNAs with 5′-overhang) (pathway to the right), which results in non-productive complexes with low affinity and low ATPase activity that are unable to signal, and eventually dissociate.