RIG-I Uses an ATPase-Powered Translocation-Throttling Mechanism for Kinetic Proofreading of RNAs and Oligomerization

Abstract

Summary RIG-I has a remarkable ability to specifically select viral 5’ppp dsRNAs for activation from a pool of cytosolic self-RNAs. The ATPase activity of RIG-I plays a role in RNA discrimination and activation, but the underlying mechanism was unclear. Using transient state kinetics, we have elucidated the ATPase-driven ‘kinetic proofreading’ mechanism of RIG-I activation and RNA discrimination, akin to DNA polymerases, ribosomes, and T-cell receptors. Even in the autoinhibited state of RIG-I, the C-terminal domain kinetically discriminates against self-RNAs by fast off-rates. ATP-binding facilitates dsRNA engagement, but interestingly makes RIG-I promiscuous, explaining the constitutive signaling by Singleton-Merton syndrome-linked mutants that bind ATP without hydrolysis. ATP hydrolysis dissociates self-RNAs faster than 5’ppp dsRNA, but more importantly, drives RIG-I oligomerization through translocation, that we show is regulated by helicase motif-IVa. RIG-I translocates directionally from dsRNA-end into stem-region and 5’ppp-end “throttles” translocation to provide a mechanism for threading and building a signaling-active oligomeric complex.

A real-time, transient kinetic study ofDrosophila melanogasterDicer-2 elucidates mechanism of termini-dependent cleavage of dsRNA

“…Similarly, in the presence of ATPgS, the observed signal enhancement may reflect that the stable intermediate observed with this analog is proximal to the Cy3 at position 18. Taken together, these kinetic studies demonstrate that dmDcr-2 utilizes the free energy of ATP hydrolysis to translocate along the sense (top) strand from 5' to 3', as observed for RIG-I (Myong et al, 2009;Devarkar 2018).…”

N6-methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I

“…The deficiency of m 6 A in virion RNA induces higher RIG-I expression, an enhanced RIG-I binding affinity and an enhanced ability to trigger the conformational change in RIG-I that corresponds to enhanced signaling to the downstream adaptor protein MAVS, activating the IRF3 and NF-κB pathways, leading to higher IFN-I. In addition, RIG-I is a 5’-triphosphate-dependent translocase, traveling from the 5’-ppp into the RNA chain to trigger oligomerization 44 , 45 , which has been shown to be hindered by internal 2’-O methylation in dsRNA 46 . Thus, it is possible that m 6 A methylation may also serve as a “brake” or “throttle” to prevent RIG-I translocation, oligomerization, and downstream signaling ( Fig.5g ).…”

RIG-I-like receptors: their regulation and roles in RNA sensing

“…The base-paired portion of the RNA forms contacts with the helicase domain in RIG-I 18,19,21 . The helicase domain has ATPase activity, and ATP hydrolysis determines the dissociation rate of RIG-I from RNA [37][38][39][40] . Stable RIG-I-RNA interaction displaces the CARDs, which in the inactive RIG-I conformation interact with parts of the helicase domain 18 .…”