Characterization of RNA driven structural changes in full length RIG-I leading to its agonism or antagonism
Justyna Sikorska, Yan Hou, Paul Chiurazzi, Tony Siu, Gretchen A. Baltus, Payal R. Sheth, David G. McLaren, Quang Truong, Craig A. Parish, Daniel F. Wyss
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.