title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854;
authors.forename: Swapnil C
authors.initials: SC
authors.lastname: Devarkar
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854.
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Miller
authors.affiliation: Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854;
authors.forename: Anand
authors.initials: A
authors.lastname: Ramanathan
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854.
authors.forename: Fuguo
authors.initials: F
authors.lastname: Jiang
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854.
authors.forename: Abdul G
authors.initials: AG
authors.lastname: Khan
authors.affiliation: Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854; patelss@rutgers.edu jmarco@cabm.rutgers.edu.
authors.forename: Smita S
authors.initials: SS
authors.lastname: Patel
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854 patelss@rutgers.edu jmarco@cabm.rutgers.edu.
authors.forename: Joseph
authors.initials: J
authors.lastname: Marcotrigiano
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D002352:Carrier Proteins / Q000378:metabolism; D018360:Crystallography, X-Ray; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D007113:Immunity, Innate; D008745:Methylation; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012315:RNA Caps / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism*; D012330:RNA, Double-Stranded; D015398:Signal Transduction
chemical_list: D002352:Carrier Proteins; D012315:RNA Caps; D012330:RNA, Double-Stranded; D006151:Guanosine; C016578:7-methylguanosine; D012313:RNA; D000255:Adenosine Triphosphate; D020365:RNA Helicases
keywords: RIG-I; capped RNA; crystal structure; innate immunity; self versus nonself