The intrinsically disordered CARDs-Helicase linker in RIG-I is a molecular gate for RNA proofreading.

The innate immune receptor RIG-I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG-I's C-terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG-I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD-chosen RNAs to bind the helicase domain, while at the same time blocking non-specific RNAs. These findings also indicate that CHL could represent a novel target for RIG-I-based therapeutics.

RIG-I recognizes metabolite-capped RNAs as signaling ligands.

The innate immune receptor RIG-I recognizes 5'-triphosphate double-stranded RNAs (5' PPP dsRNA) as pathogenic RNAs. Such RNA-ends are present in viral genomes and replication intermediates, and they activate the RIG-I signaling pathway to produce a potent interferon response essential for viral clearance. Endogenous mRNAs cap the 5' PPP-end with m7G and methylate the 2'-O-ribose to evade RIG-I, preventing aberrant immune responses deleterious to the cell. Recent studies have identified RNAs in cells capped with metabolites such as NAD+, FAD and dephosphoCoA. Whether RIG-I recognizes these metabolite-capped RNAs has not been investigated. Here, we describe a strategy to make metabolite-capped RNAs free from 5' PPP dsRNA contamination, using in vitro transcription initiated with metabolites. Mechanistic studies show that metabolite-capped RNAs have a high affinity for RIG-I, stimulating the ATPase activity at comparable levels to 5' PPP dsRNA. Cellular signaling assays show that the metabolite-capped RNAs potently stimulate the innate antiviral immune response. This demonstrates that RIG-I can tolerate diphosphate-linked, capped RNAs with bulky groups at the 5' RNA end. This novel class of RNAs that stimulate RIG-I signaling may have cellular roles in activating the interferon response and may be exploited with proper functionalities for RIG-I-related RNA therapeutics.