The autoinhibitory CARD2-Hel2i Interface of RIG-I governs RNA selection.

Anand Ramanathan,Fuguo Jiang,Matthew T Miller,Smita S Patel,Abdul G Khan,Joseph Marcotrigiano,Swapnil C Devarkar

doi:10.1093/nar/gkv1299

Anand Ramanathan, Fuguo Jiang + Show 5 more

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https://doi.org/10.1093/nar/gkv1299

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Abstract

RIG-I (Retinoic Acid Inducible Gene-I) is a cytosolic innate immune receptor that detects atypical features in viral RNAs as foreign to initiate a Type I interferon signaling response. RIG-I is present in an autoinhibited state in the cytoplasm and activated by blunt-ended double-stranded (ds)RNAs carrying a 5′ triphosphate (ppp) moiety. These features found in many pathogenic RNAs are absent in cellular RNAs due to post-transcriptional modifications of RNA ends. Although RIG-I is structurally well characterized, the mechanistic basis for RIG-I's remarkable ability to discriminate between cellular and pathogenic RNAs is not completely understood. We show that RIG-I's selectivity for blunt-ended 5′-ppp dsRNAs is ≈3000 times higher than non-blunt ended dsRNAs commonly found in cellular RNAs. Discrimination occurs at multiple stages and signaling RNAs have high affinity and ATPase turnover rate and thus a high katpase/Kd. We show that RIG-I uses its autoinhibitory CARD2-Hel2i (second CARD-helicase insertion domain) interface as a barrier to select against non-blunt ended dsRNAs. Accordingly, deletion of CARDs or point mutations in the CARD2-Hel2i interface decreases the selectivity from ≈3000 to 150 and 750, respectively. We propose that the CARD2-Hel2i interface is a ‘gate’ that prevents cellular RNAs from generating productive complexes that can signal.

Highlights

RIG-I like receptors, found in most cell types, are cytosolic sensors of viral RNAs
By carrying out studies with full-length RIG-I, RIG-I domains and interface mutant, we show that CARD2-Hel2i interface plays a key role in RNA selection by acting as a ‘gate’ to allosterically regulate both RNA affinity and ATPase activity
We studied longer dsRNAs and hairpin RNAs. Using this panel of dsRNAs of the same sequence, but different end-modifications, we studied RNA binding and ATPase activation of RIG-I, Helicase-RD lacking the caspase activation and recruitment domains (CARDs), RIG-I lacking just the first CARD, the C-terminal repressor domain (RD) and the CARD2-Hel2i interface mutant (Figure 1B)

Summary

Introduction

RIG-I like receptors, found in most cell types, are cytosolic sensors of viral RNAs. These receptors include RIG-I, MDA5 and LGP2, which are superfamily 2 RNA helicases/ATPases with the ability to recognize specificRNA features and trigger an immune response against a wide variety of commonly found RNA viruses [1,2,3,4,5]. RIG-I recognizes 5 -triphosphate (5 ppp) and 5 pp [6,7,8] on basepaired and blunt-ended double-stranded (ds)RNAs [9,10] found in many viral genomes and their replication intermediates [11,12,13,14,15,16] These features are not present in cellular RNAs because of post-transcriptional modifications and the lack of an RNA-dependent RNA polymerase in mammalian cells. Biochemical studies have measured RNA affinities and ATPase turnover rates of blunt-ended 5 ppp and 5 OH dsRNAs [19,20,21], but a systematic study of the effect of RNA-end modifications on RNA affinity, ATPase activity and signaling is lacking Such studies, for example, of the immune receptor PKR (Protein kinase R) yielded insights into cellular modifications that prevent self RNAs from activating PKR [22,23]

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