Characterization of RNA driven structural changes in full length RIG-I leading to its agonism or antagonism.

Abstract

RIG-I (retinoic acid inducible gene-I) can sense subtle differences between endogenous and viral RNA in the cytoplasm, triggering an anti-viral immune response through induction of type I interferons (IFN) and other inflammatory mediators. Multiple crystal and cryo-EM structures of RIG-I suggested a mechanism in which the C-terminal domain (CTD) is responsible for the recognition of viral RNA with a 5'-triphoshate modification, while the CARD domains serve as a trigger for downstream signaling, leading to the induction of type I IFN. However, to date contradicting conclusions have been reached around the role of ATP in the mechanism of the CARD domains ejection from RIG-I's autoinhibited state. Here we present an application of NMR spectroscopy to investigate changes induced by the binding of 5'-triphosphate and 5'-OH dsRNA, both in the presence and absence of nucleotides, to full length RIG-I with all its methionine residues selectively labeled (Met-[ϵ-13CH3]). With this approach we were able to identify residues on the CTD, helicase domain, and CARDs that served as probes to sense RNA-induced conformational changes in those respective regions. Our results were analyzed in the context of either agonistic or antagonistic RNAs, by and large supporting a mechanism proposed by the Pyle Lab in which CARD release is primarily dependent on the RNA binding event.