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 doublestranded (ds) RNAs bind to RIG-I with nearly identical K d 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′-Omethylated 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.etinoic Acid Inducible Gene-I (RIG-I) is a cytosolic innate immune receptor with the remarkable ability of distinguishing cellular self RNAs from pathogenic nonself RNAs (1, 2). RIG-I belongs to the DExH/D-box family of RNA helicases and has a multidomain architecture with three helicase domains (Hel1, Hel2, and Hel2i) located centrally, flanked by a C-terminal repressor domain (RD) and two N-terminal Caspase Activation and Recruitment Domains (CARDs) (3-7). The helicase and RD are involved in RNA recognition and binding whereas the N-terminal CARDs relay the signal to downstream factors. RIG-I is present in an inactive autoinhibited state in the absence of pathogen associated molecular pattern (PAMP) RNA ligand, but upon PAMP RNA binding, RIG-I gets activated to initiate a cell signaling response ultimately leading to Type I IFN production.RNAs carrying a 5′-triphosphate (5′ppp) moiety and bluntended double-stranded (ds)RNAs are the best characterized PAMP ligands of RIG-I, showing high-affinity binding and robust stimulation of the ATP hydrolysis activity (8-10). Although RIG-I is surrounded by cellular RNAs, they do not activate RIG-I due to posttranscriptional modification of the RNA 5′ ends. The 5′ end of many cellular RNAs like mRNAs is modified with the addition of a 7-methyl guanosine (m7G) connected by a 5′-to-5′ triphosphate bridge to the first nucleotide (C...

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

“…The N-7-methylation of the viral RNA cap by ZIKV NS5-MTase allows recognition by the translation initiation factor eIF4E and stimulation of translation into viral proteins (8). On the other hand, 2=-O-methylation of the cap is expected to mask the presence of exogenous viral RNAs from host cell sensors such as RIG-I and MDA5 (9)(10)(11), which induce the production of interferons. In addition, 2=-O-methylation of viral RNA is thought to prevent translation restriction by interferon-stimulated genes (ISGs) such as the IFIT-1 gene (12,13).…”

“…The N-7 methylation of the cap structure is essential for RNA stability and stimulates their translation into viral protein by recognizing the translation initiation factor eIF4E (8). The 2=-O-methylation of the cap structure was demonstrated to protect viral RNA from being recognized by host cell sensors such as RIG-I and MDA5 that stimulate the production of interferons (9)(10)(11). In turn, interferon-stimulated genes, such as IFIT-1, can detect miscapped RNA and restrict their translation into proteins (12,13).…”

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

“…In different eukaryotes the levels of cap-0, cap-1, and cap-2 in mRNA vary. The amount of methylation has increased during evolution (Banerjee 1980) and is implicated in immunogenicity (Daffis et al 2010;Devarkar et al 2016).…”

“…This is especially reflected in the low number of structures of proteins in complex with capped RNA (>1 nt) that have been determined to date. Currently, these structures include the human 2 ′ -O-ribose methyltransferase CMTr1 with a capped 4-mer that was produced by chemical coupling (Smietanski et al 2014), the 2 ′ -O-ribose methyltransferase of vaccinia virus with a capped 6-mer that was produced by in vitro transcription in the presence of a cap analog (Hodel et al 1998), the 2 ′ -O-ribose methyltransferase in the NS5 protein from dengue virus with an 8-mer cap-0 viral RNA that was produced using a cap analog (Zhao et al 2015), the dengue virus methyltransferase in complex with a 5 ′ -capped RNA 8-mer that was chemically synthesized (Yap et al 2010), and the innate immune receptor RIG-I that contains a chemically synthesized 24-nt-long capped hairpin RNA (Devarkar et al 2016). The high-resolution structural data available is thus confined to a small subset of the numerous enzymes and proteins that directly interact with the mRNA cap structure.…”

Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

“…For a long time, it was unclear why the 5 0 end of mRNAs would undergo such extensive methylation. Recent studies have revealed that 2 0 -O methylation of cellular RNA plays a central role in discrimination of self from non-self RNA, e.g., distinction of viral from host RNA [10,11], while reversible interconversion of N6, 2 0 -Odimethyladenosine and 2…”