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Biochemical and Structural Basis of RNA Recognition and Activation by the Innate Immune Receptor RIG-I

Anand Ramanathan
M.Sc., Bangalore University - 2004

Thesis Advisor: Smita S. Patel, Ph.D.
Graduate Program in Biochemistry & Molecular Biology

RWJ Research and School of Public Health Building
Room 258

Monday, April 29, 2013
10:30 a.m.


(RIG)-I-like receptors (RLRs) of the innate immune system are the cell’s principal detector of RNA viruses in the cytosol. RIG-I recognizes blunt-ended double-stranded (ds) RNA and 5`-triphosphate RNA as viral Pathogen Associated Molecular Patterns (PAMPs) and triggers an immune response. RIG-I contains three domains; the N-terminal signaling CAspase Recruitment Domains (CARD), the central helicase/ATPase domain and the C-terminal RNA binding repressor domain (RD). My thesis focuses on the roles of each domain in RIG-I:RNA interaction and the mechanism of activation. The structure of human RIG-I helicase-RD bound to blunt-ended dsRNA and ADP•BeF3 was determined by crystallography. The structure reveals that both helicase and RD domains are involved in dsRNA binding. The helicase-RD organizes into a capped ring around the blunt-ended dsRNA and the helicase contacts the dsRNA using newly identified contacts. In the absence of RNA, RIG-I is flexible and compacts upon RNA binding. Biochemical studies indicate a strong synergy between the helicase and RD in RNA recognition and ATP binding enhances the RNA binding affinity of RIG-I.
It is known that RIG-I is stimulated robustly by blunt-ended dsRNA and 5’-ppp dsRNA whereas RNAs containing 3’ or 5’ overhangs do not induce a strong immune response. The second part of my thesis concerns with determining the correlation between signaling efficiency, ATPase activity, and RNA binding affinities of RIG-I. My studies find that signaling RNAs bind more tightly and stimulate the ATPase activity of RIG-I to a greater extent. Blunt ended dsRNAs with 5’-ppp have the highest affinity for RIG-I and highest ATPase rate followed by blunt-ended dsRNA without 5’-ppp, whereas 3’-overhang RNAs resembling microRNAs bind more weakly with lower ATPase than blunt-ended dsRNA, but more tightly than 5’-overhang dsRNA, and RNA stems bind with the weakest affinity and stimulate the ATPase to lesser extent. Removal of RIG-I CARDs resulted in loss of this discrimination between blunt-ended and non blunt-ended dsRNAs indicating that CARDs are involved in the autoinhibition mechanism of RIG-I. Overall, my studies have provided the structural and biochemical basis for understanding the first step of immune activation to viral infection, a new perspective for understanding how other homologous RNA helicases may engage their targets, and a new role for the CARD domains as gatekeepers to enable activation of RIG-I only by PAMP RNA.

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