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

Swapnil C Devarkar1, Chen Wang2, Matthew T Miller2

  • 1Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854;

Proceedings of the National Academy of Sciences of the United States of America
|January 7, 2016
PubMed

Insights

RNA capping and 2’-O-methylation are key to evading innate immunity receptor RIG-I. This study reveals how these modifications, particularly Cap-1, prevent RIG-I activation, offering insights into immune evasion strategies.

Area of Science:

  • Immunology
  • Structural Biology
  • Molecular Biology

Background:

  • The innate immune receptor RIG-I (Retinoic Acid Inducible Gene-I) detects 5'-triphosphate (ppp) RNAs in the cytoplasm, initiating a Type I IFN response.
  • Self RNAs, like mRNAs, are typically protected from RIG-I recognition through 5' capping modifications (Cap-0 and Cap-1).

Purpose of the Study:

  • To elucidate the structural and mechanistic roles of RNA capping (Cap-0 and Cap-1) and 2'-O-methylation in evading RIG-I recognition.
  • To understand how these modifications influence RIG-I binding affinity, ATPase activity, and downstream signaling.

Main Methods:

  • X-ray crystallography was used to determine the structures of RIG-I in complex with double-stranded (ds) RNAs bearing 5'OH, 5'ppp, and Cap-0 modifications.
  • Biochemical assays were employed to measure RNA binding affinities (Kd values) and RIG-I ATPase activity.
  • Site-directed mutagenesis (H830A) was performed to investigate the role of specific residues in RIG-I-RNA interactions.

Main Results:

  • Cap-0 and 5'ppp dsRNAs bind RIG-I with similar affinities and activate its ATPase and signaling functions.
  • Single-stranded RNAs with Cap-0 or 5'ppp modifications did not bind RIG-I or induce signaling.
  • Cap-1 modifications, particularly in conjunction with m7G capping, significantly weaken RIG-I binding affinity and reduce ATPase activity, with H830 identified as critical for discrimination.
  • A single H830A mutation in RIG-I restored high-affinity binding and signaling activity to 2'-O-methylated dsRNAs.

Conclusions:

  • RIG-I's ability to accommodate the Cap-0 structure without disrupting ppp interactions is demonstrated through crystal structures.
  • The H830 residue is crucial for RIG-I's discrimination against Cap-1 modified RNAs, a key mechanism for host immune evasion.
  • m7G capping and 2'-O-methylation act synergistically to prevent RIG-I activation, highlighting the evolutionary complexity of RNA cap structures in host-pathogen interactions.
Keywords:
RIG-Icapped RNAcrystal structureinnate immunityself versus nonself

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