The innate immune system acts as the first line of defense to stimulate the hosts response against microbial and viral infections. Specific receptors of the innate immune system detect molecular patterns in the pathogen to induce the production of interferon and proinflammatory cytokines. Interferon induces the expression of hundreds of genes to establish an antiviral state and modulate adaptive immunity, further strengthening the hosts response against infection. RIG-I (Retinoic acid Inducible Gene - I) is the prototypical member of one family of receptors that plays a critical role in discriminating between viral and cellular RNA in the cytoplasm. The RIG-I family of receptors recognizes a variety of of important human viruses, including Influenza, Hepatitis C, Dengue, West Nile, Respiratory Syncytial, Reovirus, and Ebola. A major goal of our research is to understand the thermodynamic, kinetic, and structural mechanisms by which RIG-I recognizes atypical features of infecting pathogens called PAMPs (Pathogen Associated Molecular Patterns). The RIG-I protein consists of two N-terminal CAspase Recruitment Domains (CARDs), a central DExD/H RNA helicase domain followed by a C-terminal repression domain (RD). To understand synergy among the different domains of RIG-I for RNA binding and the contribution of ATP hydrolysis to RIG-I activation, my laboratory determined the structure of the RNA binding portion of human RIG-I (helicase-RD) bound to both dsRNA and a non-hydrolysable ATP analogue, yielding new insights into RIG-I activation. RNAs with 5'-triphosphate (ppp) are detected in the cytoplasm principally by RIG-I. 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 group to the 5'-end nucleotide ribose (Cap-1). We provided structural and mechanistic basis for exact roles of capping and 2'-O-methylation in evading RIG-I recognition. Surprisingly, Cap-0 and 5'ppp dsRNAs bind to RIG-I with nearly identical affinities and activate RIG-I's ATPase and cellular signaling response to similar extents. 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 without perturbing the ppp interactions. In contrast, Cap-1 modifications abrogate RIG-I signaling through a mechanism involving the H830 residue, which we have shown is crucial for discriminating between Cap-0 and Cap-1 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 elucidated the ATPase-driven kinetic proofreading mechanism of RIG-I activation and RNA discrimination, akin to DNA polymerases, ribosomes, and T cell receptors (Devarkar et al. Mol. Cell 2018). Patients with Singleton-Merten syndrome (SMS) have a rare autoimmune disease caused by a mutation (either C268F or E373A) in RIG-I that active it for signaling in the absence of infection. ATP binding facilitates dsRNA engagement but, interestingly, the mutants make RIG-I promiscuous, explaining the constitutive signaling. ATP hydrolysis dissociates self-RNAs faster than viral dsRNA but, more importantly, drives RIG-I oligomerization through translocation. RIG-I translocates directionally from the dsRNA end into the stem region, and the 5ppp end throttles translocation to provide a mechanism for threading and building a signaling-active oligomeric complex. Furthermore, using hydrogen/deuterium exchange, mechanistic models were presented for dysregulation of RIG-I proofreading by SMS mutants that ultimately result in the improper recognition of cellular RNAs (Zheng et al. Nature Commun. 2018) .
| Nabet, Barzin Y; Qiu, Yu; Shabason, Jacob E et al. (2017) Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer. Cell 170:352-366.e13 |
| Willemsen, Joschka; Wicht, Oliver; Wolanski, Julia C et al. (2017) Phosphorylation-Dependent Feedback Inhibition of RIG-I by DAPK1 Identified by Kinome-wide siRNA Screening. Mol Cell 65:403-415.e8 |
