Structural basis for m7G recognition and 2'-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I

Abstract

RNAs with 5'-triphosphate (ppp) are detected in the cytoplasm principally by the innate immune receptor Retinoic Acid Inducible Gene-I (RIG-I), whose activation triggers a Type I IFN response. It is thought that self RNAs like mRNAs are not recognized by RIG-I because 5'ppp is capped by the addition of a 7-methyl guanosine (m7G) (Cap-0) and a 2'-O-methyl (2'-OMe) group to the 5'-end nucleotide ribose (Cap-1). Here we provide structural and mechanistic basis for exact roles of capping and 2'-O-methylation in evading RIG-I recognition. Surprisingly, Cap-0 and 5'ppp double-stranded (ds) RNAs bind to RIG-I with nearly identical Kd values and activate RIG-I's ATPase and cellular signaling response to similar extents. On the other hand, Cap-0 and 5'ppp single-stranded RNAs did not bind RIG-I and are signaling inactive. Three crystal structures of RIG-I complexes with dsRNAs bearing 5'OH, 5'ppp, and Cap-0 show that RIG-I can accommodate the m7G cap in a cavity created through conformational changes in the helicase-motif IVa without perturbing the ppp interactions. In contrast, Cap-1 modifications abrogate RIG-I signaling through a mechanism involving the H830 residue, which we show is crucial for discriminating between Cap-0 and Cap-1 RNAs. Furthermore, m7G capping works synergistically with 2'-O-methylation to weaken RNA affinity by 200-fold and lower ATPase activity. Interestingly, a single H830A mutation restores both high-affinity binding and signaling activity with 2'-O-methylated dsRNAs. Our work provides new structural insights into the mechanisms of host and viral immune evasion from RIG-I, explaining the complexity of cap structures over evolution.

Keywords: RIG-I; capped RNA; crystal structure; innate immunity; self versus nonself.

Fig. 1.

K_d, ATP hydrolysis rate, and crystal structures of the RIG-I complexes with hairpin RNA containing 5′OH, 5′ppp, and Cap-0 end-modifications. (A) The ATP hydrolysis rate of RIG-I measured at 37 °C in Buffer A is plotted against increasing concentrations of 5′OH, 5′ppp, and m7G cap HP RNA. Schematic representations of the RNAs are shown. The dependencies were fit to Eq. 2 to obtain the K_d,_app and k_atpase (Table 1). (B) Overview of the crystal structure of Helicase-RD in complex with 5′OH HP RNA (PDB ID 5F9F) (Left), 5′ppp HP RNA (PDB ID 5F9H) (Center), and Cap-0 HP RNA (PDB ID 5F98) (Right).

Fig. 2.

Interactions of RIG-I with the 5′ppp and Cap-0 end-modifications. (A) Magnified view of Hel2 and RD interaction with 5′OH HP RNA (Left), 5′ppp HP RNA (Center), and Cap-0 HP RNA (Right). The Hel2 loop-helix region (664–685) in the helicase motif IVa is ordered in the presence of 5′OH and disordered (dashed line) in the presence of 5′ppp and Cap-0. (B) The RNA ligands from the six complexes in the asymmetric unit for each crystal are superimposed using the protein for the calculation. Each color represents one of the six complexes in each unit cell. (C) Magnified view of the overlaid 5′ppp and m7Gppp moiety from B. (D) Superimposition of the m7Gppp moiety from the six complexes of the asymmetric unit is shown. Highlighted are conserved contacts within 4 Å of the m7Gppp moiety. The view in Right is rotated 90° about a vertical axis. No conserved specific contacts with the m7G were observed. (E) Magnified view of the protein contacts of the 5′ppp moiety in the 5′ppp HP and Cap-0 HP RNA structures.

Fig. 3.

K_d and ATP hydrolysis rate of RNA complexes with WT and H830A RIG-I. (A and B) A magnified view of RIG-I contacts with the 2′ hydroxyl groups of the first nucleotide ribose in the crystal structure of Helicase-RD with 5′ppp HP RNA (A) and Cap-0 HP RNA (B). (C and D) The ATP hydrolysis rate of WT RIG-I is plotted against increasing concentrations of 5′ppp 2′-OMe HP RNA (C) and Cap-1 HP RNA (D). (E and F) The ATP hydrolysis rate of H830A RIG-I is plotted against increasing concentrations of 5′ppp 2′-OMe HP RNA (E) and Cap-1 HP RNA (F). The dependencies were fit to Eq. 2 to obtain the K_d,_app and k_atpase values (Table 2).

Fig. 4.

Cell-based signaling assays to measure RIG-I activation by RNAs with various 5′-end modifications. (A) Schematic representation of all of the RNA ligands used in the cell-based signaling studies (27-bp dsRNAs and 27-mer ssRNAs). (B) The luciferase signal is plotted as the IFN-β response of WT RIG-I or H830A RIG-I transfected cells stimulated with various concentration of 5′ppp dsRNA (blue bars) and Cap-0 dsRNA (red bars). (C) The luciferase signal is plotted as the IFN-β response of WT RIG-I (blue bars) or H830A RIG-I (red bars) stimulated with the indicated RNA ligand. (D) Western blot analysis of cell lysates from mock, WT RIG-I, and H830A RIG-I–transfected cells, probed with anti–RIG-I and anti–β-actin antibodies. (E) The luciferase signal is plotted as the IFN-β response of mock or WT RIG-I stimulated with the indicated RNA ligand. Signaling data were collected from quadruplicate sets, and the SEM is shown as error bars.